Systems and methods for detection and quantification of analytes

ABSTRACT

Devices, systems, and methods for detecting molecules of interest within a collected sample are described herein. In certain embodiments, self-contained sample analysis systems are disclosed, which include a reusable reader component, a disposable cartridge component, and a disposable sample collection component. In some embodiments, the reader component communicates with a remote computing device for the digital transmission of test protocols and test results. In various disclosed embodiments, the systems, components, and methods are configured to identify the presence, absence, and/or quantity of particular nucleic acids, proteins, or other analytes of interest, for example, in order to test for the presence of one or more pathogens or contaminants in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2015/049439, filed Sep. 10, 2015, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Application No. 62/049,313, filedSep. 11, 2014, the entire contents of both of which are incorporatedherein by reference. This application is also a continuation-in-part ofU.S. patent application Ser. No. 15/368,249, filed Dec. 2, 2016, whichis a continuation of U.S. patent application Ser. No. 14/954,817, filedNov. 30, 2015, now U.S. Pat. No. 9,522,397, which is a continuation ofU.S. patent application Ser. No. 14/599,372, filed Jan. 16, 2015, nowU.S. Pat. No. 9,207,244, which is a continuation of InternationalApplication No. PCT/US2014/023821, filed Mar. 11, 2014, which claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.61/776,254, filed Mar. 11, 2013. The entire disclosures of each of theabove-identified applications are incorporated herein by reference.

TECHNICAL FIELD

The present technology relates generally to the field of moleculedetection. In particular, the technology relates to microfluidicdevices, systems, and methods for detecting the presence, absence and/orquantity of one or more particular analytes within a collected sample.

BACKGROUND

Conventional technologies for identifying the presence, absence and/orquantity of nucleic acids, proteins, and/or other molecules of interestwithin a sample often require expensive laboratory equipment and theexpertise of highly-trained medical professionals. Consequently, suchanalyses are typically performed within laboratories or medicalfacilities. Such molecule detection can be important, for example, todetect the presence of pathogens, disease, contamination, overdoses, andpoisonings within a person or other animal or within the environment.Unfortunately, today, individuals may face long waits before the propertests can be performed and before the results can be generated andanalyzed. Due to the long waits and the inconvenience of traveling to alaboratory or medical facility, illnesses and contaminations oftenspread and may cause substantial harm before the presence of saidillness or contamination is even identified.

SUMMARY

There is a significant need for improved molecule detection andquantification technologies. There is a need for devices that can detectmolecules of interest in less time and with less technical expertisethan the conventional devices used today. There is a need for moleculedetection technologies that can be utilized by consumers in non-clinicalsettings, for example, in schools, places of employment, and in thehome. There is also a need for molecule detection technologies that canbe used by consumers upon entering a pharmacy or healthcare facility,and which can generate results quickly so that results are available bythe time the consumer talks with a pharmacist or healthcarepractitioner. There is also a need for consumer-targeted moleculedetection devices configured to minimize biohazard risks. Variousembodiments disclosed herein may fulfill one or more of these needs.

One aspect of the disclosure is directed to a system for detectingmolecules. In various embodiments, the system includes a cartridgedevice, a reader device removably coupled to the cartridge device, and asample collection device. In some embodiments, the cartridge deviceincludes: a cartridge housing having internal barriers defining aplurality of reservoirs, an analysis channel, and an input tunnel; and acircuit board coupled to or disposed within the cartridge housing, thecircuit board forming a wall of the analysis channel and having aplurality of sensors disposed within a portion of the analysis channel.In some embodiments, the reader device includes: a magnet aligned withthe sensor; a circuit electrically coupled to the sensor; and aprocessor having memory with instructions stored thereon. In suchembodiments, the reader device also includes a reader housing in whichthe magnet, circuit, and processor are located, the reader housingdefining a dock which receives at least a portion of the sample analysiscartridge. In some embodiments, the sample collection device is sized tofit at least partially within the input tunnel. Additionally, in someembodiments, the molecule detection system also includes a sonicationcomponent electrically coupled to the circuit and aligned with a firstof the plurality of reservoirs. The sonication component may form acomponent of the cartridge device or the reader device and can becomprised partially or wholly of a piezoelectric transducer.

Another aspect of the disclosure is directed to a sample analysiscartridge. In some embodiments, the cartridge includes a housing and acircuit board disposed on, under, or within the housing. In someembodiments, the housing has internal barriers defining a plurality ofreservoirs, an analysis channel, and an input tunnel. The plurality ofreservoirs includes a first reservoir at least partially filled with afirst liquid volume comprising sample preparation reagents and anotherreservoir at least partially filled with a liquid volume comprising achemical substrate. In some embodiments, the plurality of reservoirsadditionally includes a reservoir at least partially filled with aliquid volume comprising a wash solution. In certain embodiments, theinput tunnel extends from an aperture at a surface of the housing to thefirst reservoir and each of the plurality of reservoirs is, at least attimes, in fluid communication with the analysis channel. In certainembodiments, the circuit board includes a plurality of sensors alignedwith a portion of the analysis channel.

In some such embodiments, the sample preparation reagents include aplurality of magnetic particles having surface-bound affinity molecules,a plurality of detector agents, and a plurality of agents to facilitateaccess to the target analyte and binding between the target analyte andthe surface-bound affinity molecules and binding between the targetanalyte and the detector agents. In other embodiments, the cartridgealso includes a membrane disposed between the input tunnel and the firstreservoir. The membrane of some such embodiments dry-stores one or moreof the magnetic particles, the detector agents, the binding agents,and/or other sample preparation reagents, storing said reagents untilthe membrane is ruptured, at which point, said reagents enter the firstreservoir. The membrane of other embodiments dry-stores a plurality ofcompetitive binding agents, each competitive binding agent including apre-bound target analyte bound to a signaling agent. In suchembodiments, the sample preparation reagents include a plurality ofmagnetic particles having surface-bound affinity molecules and aplurality of agents to facilitate access to the target analyte and tofacilitate binding of the surface-bound affinity molecules to the targetanalyte or the competitive binding agent. In some such embodiments, thesample preparation reagents are stored, prior to membrane rupture,within the first reservoir; in other embodiments, one or more of thesample preparation reagents are stored, prior to membrane rupture, onthe membrane.

In various embodiments, the plurality of magnetic particles may includemagnetic particles of two or more sizes, each size having a differentsurface-bound affinity molecule such that each size binds to a differenttarget analyte.

In some embodiments of a sample analysis cartridge, the cartridgeincludes a plurality of valves corresponding with the plurality ofreservoirs with one valve positioned at each intersection between one ofthe plurality of reservoirs and the analysis channel. In some suchembodiments, each of the plurality of valves is phase-changeable uponthe application of heat, and the circuit board includes a plurality ofvias aligned with (e.g., disposed directly above or below) the pluralityof valves; such vias are physically coupled to a heating element. Insome embodiments, the sample analysis cartridge further includes anabsorbant material disposed at a downstream end of the analysis channel.

In various embodiments of the cartridge, the housing includes a covercomponent, an internal component, and a base component coupled togetherto form a fixed structure. In some such embodiments, the cover componentis disposed on a first side of the internal component, the basecomponent is disposed on a second side of the internal component, andthe circuit board is positioned between the internal component and thebase component. Features of the cover component and the first side ofthe internal component may together define the input tunnel and theplurality of reservoirs, and features of the second side of the internalcomponent and the circuit board may together define the analysischannel.

An additional aspect of the disclosure is directed to a sample analysisreader. In various embodiments, the reader includes a magnetic fieldgenerator, a circuit having a cartridge detection unit, a processorhaving memory with instructions stored thereon, and a housing with adock for coupling to a sample analysis cartridge. In certainembodiments, when the sample analysis reader is coupled to the sampleanalysis cartridge, the magnetic field created by the magnetic fieldgenerator is substantially aligned with a sensor of the sample analysiscartridge, and the circuit is electrically coupled to the sensor of thesample analysis cartridge. In various embodiments, the sample analysisreader interchangeably couples to a plurality of sample analysiscartridges.

In some embodiments, the reader also includes a sonication componentelectrically coupled to the circuit. In such embodiments, when a sampleanalysis reader is coupled to the sample analysis cartridge, thesonication component is aligned with a sample preparation reservoir inthe sample analysis cartridge.

In some embodiments of the sample analysis reader, the magnetic fieldgenerator includes a plurality of magnet field generators, and when thesample analysis reader is coupled to the sample analysis cartridge, theplurality of magnet field generators are aligned with a plurality ofsensors lying on a plane of the sample analysis cartridge with eachmagnetic field generator configured to produce a magnetic field of adifferent strength. Such a configuration creates a magnetic fieldgradient within the analysis channel of the sample analysis cartridge.In some embodiments, the plurality of magnetic field generators areformed of a plurality of permanent magnets, each disposed at a differentdepth relative to the plane of the sensors. In other embodiments, themagnetic field gradient may be formed, for example, using a plurality ofpermanent magnets of increasing size or a plurality of inductors ofincreasing size or increasing numbers of coils.

In some embodiments of the reader, the sonication component is apiezoelectric component electrically coupled to the processor, and thepiezoelectric component is positioned to transduce a mechanical event ormechanical change within the reservoir into an electrical signal. Insuch embodiments, a processor and/or circuitry electrically coupled tothe piezoelectric component is configured to receive and interpret theelectrical signal. This mechanical event in the reservoir can betransduced in the form of detected pressure applied to the piezoelectriccomponent through flex in the sample preparation reservoir of the sampleanalysis cartridge upon entry of a sample collection device.Alternatively, a change in the mechanical load or mass above thepiezoelectric component can cause a shift in the resonance frequency ofthe piezoelectric component that is detectable and/or quantifiable bythe processor and/or circuitry. In other embodiments, the piezoelectriccomponent and connected processor and/or circuitry quantify variation inthe reflected wave of a pulse emitted from the piezoelectric component.In some such embodiments, the processor and/or circuitry is programmedwith a threshold value for such variation in the reflected wave, thethreshold set to distinguish between a state of having no collectiondevice within the reservoir versus a collection device inserted state.In yet another example of the piezoelectric component transducing amechanical event or mechanical change within the reservoir into anelectrical signal, the piezoelectric component is configured to detect asound wave such as the sound wave corresponding with a clicking that isactuated by mechanical parts of the sample collection device interactingwith features of the input tunnel or reservoir.

In some embodiments of the sample analysis reader, the processor isconfigured to execute the instructions stored in memory, which whenexecuted, cause the processor to perform a method. The method of certainembodiments includes identifying a proper test protocol for the coupledsample analysis cartridge based at least in part on cartridgeidentification information received from the circuit, and executing theproper test protocol. In some embodiments, executing the proper testprotocol includes: stimulating the piezoelectric component to generate atest signal within the sample preparation reservoir and to detect areturn signal, receiving detection signals from the piezoelectriccomponent, the detection signals including the return signal and aresonance of the piezoelectric component, detecting entry of a samplecollection device into the sample preparation reservoir based at leastin part on a change in the return signal and/or a shift in the resonanceof the piezoelectric component, and initiating a sonication protocol forthe sonication component to mix reagents and sample particles within aliquid disposed within the sample preparation reservoir, wherein mixingfacilitates hybridization of at least some of the reagents with thesample particles.

In some embodiments, the method performed by the processor whenexecuting the proper test protocol additionally or alternativelyincludes receiving via the circuit, detection signals generated by thesensor of the sample analysis cartridge, and processing the detectionsignals. The method may also include transmitting data based at least inpart on the detection signals to a mobile computing device or displaydevice.

A further aspect of the disclosure is directed to a specialized computerfor non-clinical disease detection. The specialized computer of variousembodiments includes both hardware and software. For example, in someembodiments, the computer includes a dock or port for engaging at leasta portion of a disease detection cartridge, the dock positioned on orwithin the computer. The computer of various embodiments also includes:circuitry for detecting signals generated from an oxidation reactionoccurring within the disease detection cartridge, and a processor havingmemory with instructions stored thereon. Upon engagement with thedisease detection cartridge, the processor executes the instructions,which in certain embodiments, causes the processor to perform a methodthat includes: detecting a classification of the disease detectioncartridge from signals received from the circuitry, initiating a testingprotocol specific to the classification, and generating diseasedetection results specific to the classification in less than thirtyminutes. The method may further include transmitting the diseasedetection results to a remote computing device for further processing,display, and/or storage. In certain embodiments, the computer is lessthan 30 cm in height, less than 30 cm in width, and less than 30 cm inlength. In certain embodiments, the computer is intended for use bynon-trained consumers in home, office, or school settings.

One aspect of the disclosure is directed to a self-contained analytedetection kit, which securely stores, during and after analytedetection, all collected sample and all liquids needed to detect aspecific analyte. In various embodiments, the kit includes aone-time-use sample collection device; and a one-time-use detectionunit. The detection unit includes an input tunnel sized to securely andpermanently receive the sample collection device, and a plurality ofcompartments, which separately and securely store reagents, a washmedia, and a substrate. In some embodiments, the input tunnel extendsfrom an aperture on a surface of the detection unit to an entryway of afirst compartment holding the reagents. In some embodiments, prior toinsertion of the sample collection device, a selectively breakablemembrane covers the entryway of the first compartment to block flow ofliquid and/or reagents into the input tunnel. In some embodiments,complementary locking features are disposed on the sample collectiondevice and in the input tunnel to restrict movement of the samplecollection device relative to the detection unit upon insertion of thesample collection device into the input tunnel. Moreover, in someembodiments, the sample collection device and input tunnel are sized toform a liquid-tight seal as the sample collection device advances intothe input tunnel.

Still a further aspect of the disclosed technology is directed to amethod for detecting a disease without a healthcare provider ortechnician present. In some embodiments, such a method includes: rubbingan internal passage of a user's nose with a swab to collect a sample,placing a cartridge, which houses all reagents and substrates needed toperform a disease-detection testing protocol, into or onto a specializedcomputer configured to detect the cartridge, and inserting the swab intothe cartridge such that the swab locks into place within the cartridgeand cannot be removed. In various embodiments, the specialized computersenses the insertion of the swab and initiates a testing protocol. Insome such embodiments, the specialized computer detects the presence orabsence of a particular disease within the sample via the testingprotocol in less than 30 minutes. The method may also include readingresults of the test from a remote computing device, after the resultsare transmitted from the specialized computer to the remote computingdevice via a wired or wireless communication connection.

An additional aspect of the disclosure is directed to a method fordetecting the presence, absence, and/or quantity of a target analytewithin a sample. The method of various embodiments includes: loading acartridge into or onto an analyte reader, wherein the cartridge has aplurality of reservoirs, including a first reservoir filled at leastpartially with reagents, a reservoir filled at least partially with asubstrate, and optionally, another reservoir filled at least partiallywith a wash solution; removing a sample collection device from a sterilepackage; contacting a specimen with a tip of the sample collectiondevice to collect a sample; and inserting the sample collection deviceinto the cartridge until at least the tip enters the first reservoir. Incertain embodiments, inserting the tip of the sample collection deviceinto the first reservoir activates the analyte reader, causing asonication device within the analyte reader to perform a sonicationprotocol to mix the sample collected by the sample collection devicewith the reagents in the first reservoir. Additionally or alternatively,inserting the tip into the first reservoir causes a series of heatingelements to sequentially melt a series of valves positioned within ornear the plurality of reservoirs, thereby sequentially releasing thecontents of the plurality of reservoirs into an analysis zone foranalysis by the analyte reader. In some such embodiments, inserting thetip of the sample collection device into the cartridge involvesadvancing the sample collection device into an input tunnel of thecartridge until: the tip of the sample collection device breaks amembrane barrier disposed at a distal end of the input tunnel, the tipenters the first reservoir, and the sample collection device locks intofixed engagement with the cartridge with a liquid-tight seal formedbetween the sample collection device and the input tunnel.

Another aspect of the disclosure is directed to computerized methods ofdetecting the presence, absence, and/or quantity of target analyteswithin a sample. For example, in some embodiments, a method performed bya computerized analyte reader includes: detecting the presence of acartridge loaded into or onto the analyte reader, detectingidentification information associated with the cartridge, andidentifying a proper test protocol for the cartridge based at least inpart on the identification information. In some embodiments, thecomputerized method additionally or alternatively includes: detecting asample collection device inserted into a first reservoir of thecartridge, initiating a sonication protocol upon sample collectiondevice insertion in order to mix a plurality of reagents, a plurality ofmagnetic particles, a plurality of detector agents or competitivebinding agents, and a plurality sample particles within the firstreservoir. In some such embodiments, the plurality of magnetic particlesincludes at least: a plurality of large magnetic particles each having afirst surface affinity molecule on its surface configured to bind to afirst target analyte, and a plurality of small magnetic particles eachhaving a second surface affinity molecule on its surface configured tobind to a second target analyte. Upon mixing, for example, via thesonication protocol, if the first and/or the second target analyte ispresent, hybridization occurs. In some such embodiments, particularlyembodiments with detector agents, the resulting mixture includes aplurality of sandwich complexes, each formed of a target analyte boundto both a surface affinity molecule on a surface of a magnetic particleand a detector agent. In other embodiments, particularly, embodimentswith a competitive binding agent, the resulting mixture includesmolecule complexes each formed of a target analyte bound only to asurface affinity molecule on a surface of a magnetic particle.

In some embodiments, the method also includes stimulating a firstheating element such that a first valve within the cartridge melts andthe mixture flows out of the sample preparation reservoir into ananalysis channel. In various embodiments, the mixture is suspended in asolution, and the solution acts as a transport medium transporting themixture from the first reservoir into the analysis channel towards adownstream absorbent material via capillary action. Within the analysischannel, the magnetic particles of the mixture localize over a pluralityof magnets or other magnetic field generators within a portion of theanalysis channel; the magnetic particles thereby form a plurality oflocalized samples. In such embodiments, the magnetic particles localizebased on size and strength such that the large magnetic particleslocalize within a smaller upstream magnetic field and the small magneticparticles localize within a larger downstream magnetic field. The methodof some embodiments also includes stimulating a second heating elementsuch that a second valve within the cartridge melts and a wash solutionflows out of a second reservoir into the analysis channel with the washsolution removing, from the plurality of localized samples, detectoragents and/or competitive binding agents that are not indirectly boundto magnetic particles. The method of some embodiments further includesstimulating a third heating element such that a third valve within thecartridge melts and a solution of substrates flows out of a thirdreservoir into the analysis channel. In some embodiments, the detectoragents and competitive binding agents include oxidizing enzymes whichoxidize the substrate.

The computerized method may further include: detecting a first signal ata first recording sensor located within the smaller magnetic field,wherein at least a portion of the first signal is caused by theoxidation of the substrate; detecting a second signal at a secondrecording sensor located near the larger magnetic field, wherein atleast a portion of the second signal is caused by the oxidation of thesubstrate; detecting a reference signal at a reference sensor;calculating a first resultant signal, for example, by subtracting thereference signal from the first signal to eliminate noise; processingand analyzing the first resultant signal to identify the presence and/orquantity of the first target analyte; calculating a second resultantsignal, for example, by subtracting the reference signal from the secondsignal to eliminate noise; and processing and analyzing the secondresultant signal to identify the presence and/or quantity of the secondtarget analyte. In some embodiments, the method also includestransmitting signals indicative of a test result to a mobile computingdevice.

In some such embodiments, the first resultant signal is proportional toa quantity of the first target analyte present within the localizedsamples and the second resultant signal is proportional to a quantity ofthe second target analyte present within the localized samples. In otherembodiments, the first and second resultant signals are indirectlyproportional to a quantity of first and second target analytes presentin the sample. In other embodiments, the first signal is indirectlyproportional to the quantity of first analyte and the second signal isdirectly proportional to the quantity of second target analyte, or viceversa.

In other embodiments of a computerized method for detecting thepresence, absence, and/or quantity of target analytes within a sample,the first reservoir only includes one size of magnetic particles andonly one magnet or other magnetic field generator is provided in or nearthe analysis channel. In such embodiments, the method allows for thedetection of the presence, absence, and/or quantity of a single targetanalyte.

In other embodiments of the computerized method, three or more sizes ofmagnetic particles are present in the first reservoir and an equalnumber of three or more magnetic field generators are provided in ornear the analysis channel. In such a manner, a single device and singlemethod may be employed to test for the presence of a plurality ofanalytes within a sample. Any number of particle sizes and magneticfield strengths can be utilized to create a 1-to-1 mapping betweensensor signal and analyte target concentration whether that signal bedirectly or indirectly proportional to quantity of target analyte. Insuch embodiments, the number of magnetic fields is equal to the numberof sensors and the number of unique magnetic particle populations, whichare both equal to the number of different target analytes the system isconfigured to detect. Such methods and devices may be used, for example,to determine: from which illness, among many, a person is suffering; towhich drug or poison, among many, a person is adversely reacting; orwhich chemical, among many, has contaminated the water. Other examplesinclude quantifying the concentrations of various vitamins, hormones,proteins, or other analytes of interest within one's body.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are described below with reference to theaccompanying drawings, wherein like numerals denote like elements. Inthe drawings:

FIGS. 1A-1D provide schematic depictions of molecules and reactionsfound within one embodiment of the presently disclosed analyte detectionsystem.

FIGS. 2A-2B provide schematic depictions of molecules and reactionsfound within another embodiment of the presently disclosed analytedetection system.

FIGS. 3A-3B depict a side view and perspective view, respectively, ofone embodiment of a sample collection device.

FIGS. 3C-3D depict a perspective view and side view, respectively, ofthe collection head provided in the sample collection device embodimentof FIGS. 3A-3B.

FIG. 4A depicts a side view of another embodiment of a sample collectiondevice.

FIG. 4B depicts a perspective view of the sample collection device ofFIG. 4A.

FIG. 5 depicts a functional block diagram of one embodiment of a samplecollection device.

FIG. 6 depicts a side view of another embodiment of a sample collectiondevice.

FIG. 7A depicts a perspective view of one embodiment of an assembledcartridge device.

FIG. 7B depicts a perspective view of components forming the cartridgedevice of FIG. 7A in a disassembled configuration.

FIG. 8 depicts an exploded view of another embodiment of a cartridgedevice.

FIGS. 9A-9C depict exploded, semi-exploded, and non-exploded perspectiveviews of another cartridge device embodiment.

FIG. 10A depicts a top view of the cartridge device embodiment of FIG.8.

FIG. 10B depicts a partial perspective view of the cartridge device fromFIG. 8.

FIGS. 11A-11B depict a top view and perspective view, respectively, ofan internal component and a circuit board component found in oneembodiment of a cartridge device.

FIG. 11C depicts a partial view of the internal component of FIG. 11Azoomed to highlight the features of the reservoirs in the particularembodiment.

FIGS. 12A-12B depict a top view and a side view, respectively, of thecartridge device embodiment of FIG. 8 having the sample collectiondevice embodiment of FIGS. 4A-4B disposed therein.

FIGS. 13A-13B depict a top view and a perspective view of one embodimentof a sample preparation reservoir schematically represented in isolationfrom the remainder of a cartridge.

FIG. 14 depicts a functional block diagram of one embodiment of an inputtunnel.

FIGS. 15A-15C depict a top view, side view, and perspective view,respectively, of another embodiment of an input tunnel.

FIG. 16A depicts a top view of one embodiment of an input tunnel whereinone embodiment of a sample collection device is disposed therein in alocked configuration.

FIGS. 16B-16C depict partial views of the input tunnel and samplecollection device of FIG. 16A zoomed to highlight the embodiment'slocking features and sealing features, respectively.

FIGS. 17A-17I depict cross-sectional views of various embodiments of amicrofluidic analysis channel.

FIGS. 18A-18B depict a top view and a bottom view, respectively, of thecircuit board component embodiment of the cartridge embodiment of FIGS.7A-7B.

FIG. 19 depicts a cross-sectional view of a first reservoir from thecartridge embodiment of FIG. 8.

FIGS. 20A-20B each depicts valves positioned within one embodiment of acartridge.

FIG. 21 schematically represents one embodiment of a reader device.

FIG. 22 depicts an exploded view of one embodiment of a reader device.

FIGS. 23A-23C schematically represent a sonicator engaged in variousstates of an automatic detection and automatic start protocol.

FIG. 24 depicts a schematic diagram of one embodiment of a valvefeedback system.

FIG. 25 depicts a partial view of one embodiment of a reader devicehaving a valve feedback system.

FIGS. 26A-26C depict various views of the reader device embodiment ofFIG. 22 in various stages of engagement with the cartridge deviceembodiment of FIGS. 7A-7B.

FIGS. 27A-27B provide a side view and cross-sectional view of anotherembodiment of a reader device coupled to another embodiment of acartridge device.

FIG. 28A depicts various components comprising one embodiment of atarget analyte detection system.

FIG. 28B depicts the target analyte detection system of FIG. 28A withthe various components coupled together and in use.

FIG. 29A depicts another embodiment of a reader device.

FIG. 29B depicts the reader device of FIG. 29A directly coupled to aremote computing device.

FIG. 30 depicts another embodiment of a reader device.

FIG. 31 provides a schematic diagram of one embodiment of an analytedetection system.

FIG. 32 provides a flowchart of one embodiment of a method for detectingthe presence, absence, and/or quantity of a target analyte in a sample.

FIGS. 33-37 each depict an embodiment of a graphical user interfacegenerated by a remote computing device that forms a portion of someembodiments of a target analyte detection system.

FIGS. 38-45 depict experimental results from various experimentsperformed using embodiments of the system or components of the systemdescribed herein.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form part of the present disclosure. Theembodiments described in the drawings and description are intended to beexemplary and not limiting. As used herein, the term “exemplary” means“serving as an example or illustration” and should not necessarily beconstrued as preferred or advantageous over other embodiments. Otherembodiments may be utilized and modifications may be made withoutdeparting from the spirit or the scope of the subject matter presentedherein. Aspects of the disclosure, as described and illustrated herein,can be arranged, combined, and designed in a variety of differentconfigurations, all of which are explicitly contemplated and form partof this disclosure.

Unless otherwise defined, each technical or scientific term used hereinhas the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. In accordance with the claimsthat follow and the disclosure provided herein, the following terms aredefined with the following meanings, unless explicitly stated otherwise.

The term “about” or “approximately,” when used before a numericaldesignation or range (e.g., pressure or dimensions), indicatesapproximations which may vary by (+) or (−) 5%, 1% or 0.1%.

As used in the specification and claims, the singular form “a”, “an” and“the” include both singular and plural references unless the contextclearly dictates otherwise. For example, the term “a molecule” mayinclude, and is contemplated to include, a plurality of molecules. Attimes, the claims and disclosure may include terms such as “aplurality,” “one or more,” or “at least one;” however, the absence ofsuch terms is not intended to mean, and should not be interpreted tomean, that a plurality is not conceived.

As used herein, the term “comprising” or “comprises” is intended to meanthat the devices, systems, and methods include the recited elements, andmay additionally include any other elements. “Consisting essentially of”shall mean that the devices, systems, and methods include the recitedelements and exclude other elements of essential significance to thecombination for the stated purpose. Thus, a device or method consistingessentially of the elements as defined herein would not exclude othermaterials or steps that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanthat the devices, systems, and methods include the recited elements andexclude anything more than a trivial or inconsequential element or step.Embodiments defined by each of these transitional terms are within thescope of this disclosure.

As used herein, an “antibody” includes whole antibodies (monoclonalantibodies and polyclonal antibodies) and any antigen binding fragmentor a single chain thereof. Thus the term “antibody” includes any proteinor peptide containing molecule that comprises at least a portion of animmunoglobulin molecule having biological activity of binding to theantigen. Examples of such may comprise a complementarity determiningregion (CDR) of a heavy or light chain or a ligand binding portionthereof, a heavy chain or light chain variable region, a heavy chain orlight chain constant region, a framework (FR) region, or any portionthereof, or at least one portion of a binding protein. The terms“polyclonal antibody” or “polyclonal antibody composition” as usedherein refer to a preparation of antibodies that are derived fromdifferent B-cell lines. A polyclonal antibody composition comprises amixture of immunoglobulin molecules secreted against a specific antigen,recognizing the same or different epitopes of the antigen, or againstdifferent antigens. The terms “monoclonal antibody” or “monoclonalantibody composition” as used herein refer to a preparation of antibodymolecules of single molecular composition. A monoclonal antibodycomposition displays a single binding specificity and affinity for aparticular epitope. A “monoclonal antibody mixture” or an “oligoclonalcocktail” refers to a mixture or combination of multiple monoclonalantibodies, each of which monoclonal antibodies can specificallyrecognize and bind the same antigen, the same or different epitopes ofthe antigen, or different antigens.

Antibodies generally comprise two heavy chain polypeptides and two lightchain polypeptides, though single domain antibodies having one heavychain and one light chain, and heavy chain antibodies devoid of lightchains are also contemplated. There are five types of heavy chains,called alpha, delta, epsilon, gamma and mu, based on the amino acidsequence of the heavy chain constant domain. These different types ofheavy chains give rise to five classes of antibodies, IgA (includingIgA₁ and IgA₂), IgD, IgE, IgG and IgM, respectively, including foursubclasses of IgG, namely IgG₁, IgG₂, IgG₃ and IgG₄. There are also twotypes of light chains, called kappa (κ) or lambda (2) based on the aminoacid sequence of the constant domains. A full-length antibody includes aconstant domain and a variable domain.

The constant domains are not involved directly in binding the antibodyto an antigen but are involved in the effector functions (ADCC,complement binding, and CDC). Human constant domains are described indetail by Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991), and by Bruggemann et al. (1987) J. Exp. Med. 166: 1351-1361;Love et al. (1989) Methods Enzymol. 178: 515-527. Other useful constantdomains are the constant domains of the antibodies obtainable from thehybridoma cell lines deposited with depositories like Deutsche Sammlungvon Mikroorganismen and Zellkulturen GmbH (DSMZ) or American TypeCulture Collection (ATCC).

Each of the heavy chain and light chain sequences of an antibody, orantigen binding fragment thereof, includes a variable domain with threecomplementarity determining regions (CDRs) as well as non-CDR frameworkregions (FRs). The terms “heavy chain” and “light chain,” as usedherein, mean the heavy chain variable domain and the light chainvariable domain, respectively, unless otherwise noted. Variable regionsand CDRs in an antibody sequence can be identified (i) according togeneral rules that have been developed in the art or (ii) by aligningthe sequences against a database of known variable regions. Methods foridentifying these regions are described in Kontermann and Dubel, eds.,Antibody Engineering, Springer, New York, N.Y., 2001, and Dinarello etal., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken,N.J., 2000. Databases of antibody sequences are described in and can beaccessed through “The Kabatman” database at bioinf.org.uk/abs(maintained by A. C. Martin in the Department of Biochemistry &Molecular Biology University College London, London, England) and VBASE2at vbase2.org, as described in Retter et al. (2005) Nucl. Acids Res. 33(Database issue): D671-D674. The “Kabatman” database web site alsoincludes general rules of thumb for identifying CDRs. The term “CDR,” asused herein, is as defined in Kabat et al., Sequences of ImmunologicalInterest, 5^(th) ed., U.S. Department of Health and Human Services,1991, unless otherwise indicated.

As used herein, a primary antibody is an antibody that has specificityto, and binds to, an epitope of the target analyte. As used herein, asecondary antibody is an antibody that has specificity to, and binds to,a primary antibody.

As used herein, the terms “signaling agent” and “label” may be usedinterchangeably and refer to a directly or indirectly detectablecompound or composition that is conjugated directly or indirectly to thecomposition to be detected. An antibody in a formulation or in acoformulation with other coformulated antibodies can be labeled tofacilitate detection or stability analysis. The term also includessequences conjugated to the polynucleotide that will provide a signalupon expression of the inserted sequences, such as green fluorescentprotein (GFP) and the like. The label may be detectable by itself (e.g.radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable. The labels can be suitablefor small scale detection or more suitable for high-throughputscreening. As such, suitable labels include, but are not limited toradioisotopes, fluorochromes, chemiluminescent compounds, dyes, andproteins, including enzymes. The label may be simply detected or it maybe quantified. A response that is simply detected generally comprises aresponse whose existence merely is confirmed, whereas a response that isquantified generally comprises a response having a quantifiable (e.g.,numerically reportable) value such as an intensity, polarization, and/orother property. In luminescence or fluorescence assays, the detectableresponse may be generated directly using a luminophore or fluorophoreassociated with an assay component actually involved in binding, orindirectly using a luminophore or fluorophore associated with another(e.g., reporter or indicator) component.

Examples of luminescent labels that produce signals include, but are notlimited to bioluminescence and chemiluminescence. Detectableluminescence response generally comprises a change in, or an occurrenceof, a luminescence signal. Suitable methods and luminophores forluminescently labeling assay components are known in the art anddescribed for example in Haugland, Richard P. (1996) Handbook ofFluorescent Probes and Research Chemicals (6^(th) ed.). Examples ofluminescent probes include, but are not limited to, aequorin andluciferases.

Examples of suitable fluorescent labels include, but are not limited to,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, and Texas Red. Other suitable optical dyes aredescribed in the Haugland, Richard P. (1996) Handbook of FluorescentProbes and Research Chemicals (6^(th) ed.).

In another aspect, the fluorescent label is functionalized to facilitatecovalent attachment to a cellular component present in or on the surfaceof the cell or tissue such as a cell surface marker. Suitable functionalgroups, including, but not are limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to attach the fluorescent label to asecond molecule. The choice of the functional group of the fluorescentlabel will depend on the site of attachment to either a linker, theagent, the marker, or the second labeling agent.

Various devices, systems, kits, and methods disclosed herein areintended to isolate, tag, and detect a target analyte within a sampletaken from a specimen. In certain embodiments, chemical reactions areemployed to enable such detection. Exemplary chemical reactions arediscussed below and depicted in FIGS. 1A-1D, 2A and 2B.

The Reactants and the Reactions

In some embodiments, a target analyte 110 a, 110 b is added to asolution of sample preparation reagents, as shown in FIGS. 1A and 1B.This target analyte may be any molecule such as a nucleic acid, protein,small molecule, or heavy metal. The sample preparation reagents at leastinclude magnetic microbeads or nanoparticles 120 a, 120 b (referred toherein as “magnetic particles”). In some embodiments, the magneticparticles are formed of an iron core (Fe₂O₃) or other ferromagneticmetal coated or otherwise surrounded by a gold shell. In someembodiments, the magnetic particles have a radius of approximately 100nanometers (nm), approximately 5000 nm, or any value therebetween. Forexample, in some embodiments, one or more of the magnetic particlepopulations have a radius of approximately 100-1000 nm. In otherembodiments, one or more of the magnetic particle populations have aradius of approximately 1000-5000 nm. In some embodiments, the magneticparticles have a radius of approximately 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000,3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200,4300, 4400, 4500, 4600, 4700, 4800, 4900, and/or 5000 nm and/or anyranges in between, e.g., 100 to 1600 nm or 200 to 1600 nm, or 300 to4900 nm. In some embodiments, a plurality of populations of magneticparticles are provided, each population having a size that is unique tothe other populations present in the solution.

In various embodiments, each magnetic particle 120 a, 120 b has anaffinity molecule 130 a, 130 b bound to its surface. As used herein, theaffinity molecule may be any suitable molecule or moiety that can bindto or capture a target molecule. Non-limiting examples of affinitymolecules include antibodies (including single chain, multi-chainantibodies, diabodies, humanized antibodies, etc.), antibody fragmentswith affinity, ligands, polypeptide or protein molecules and moietieswith binding affinity for substrates, nucleic acid molecules (e.g.,aptamers), other molecules with binding affinity, and the like. FIGS. 1Aand 1B depict an antibody 130 a and a nucleic acid probe 130 b, althoughany suitable affinity molecule could be used, including a nucleic acidaptamer or other binding protein or molecule.

In some embodiments, the sample preparation reagents also include adetector agent 140 a, 140 b, such as, for example, an antibody 160 aconjugated to a signaling agent 150 a or a labeled nucleic acid probe160 b bound to a signaling agent 150 b. In some embodiments, theantibody of the detector agent is a primary antibody with uniquespecificity to the target analyte. In other embodiments, the antibody ofthe detector agent is a secondary antibody with specificity to a primaryantibody. In such embodiments, the primary antibody may also beprovided. In some embodiments, the use of a secondary antibodyfacilitates binding of the signaling agent 150. The detector agents 140of various embodiments each include a signaling agent 150, which is adetectable label, such as, for example, an oxidizing enzyme or othersignaling enzyme, methylene blue or other electrochemically responsivetag, or a fluorescent tag such as ethidium bromide, fluorescein, greenfluorescent protein, or other fluorophore.

In embodiments that include detector agents 140, the various reagentslisted above may hybridize together to form sandwich complexes.Exemplary sandwich complexes 100 a, 100 b are illustrated in FIGS. 1Cand 1D. Each sandwich complex is formed of: (1) a magnetic particle 120a, 120 b having a surface-bound affinity molecule 130 a, 130 b, (2) atarget analyte 110 a, 110 b, and (3) a detector agent 140 a, 140 b. Theexemplary sandwich complex 100 a of FIG. 1C uses antibodies as affinitymolecules, and the target analyte is a protein or small molecule ofinterest. The exemplary sandwich complex 100 b of FIG. 1D uses nucleicacid probes designed to capture a particular sequence of nucleic acids.

In various embodiments, the signaling agent 150 is an oxidizing enzymesuch as, for example, horseradish peroxidase (HRP) or soybeanperoxidase. In such embodiments, the enzyme induces an oxidationreaction to occur at an electrochemical cell when in the presence of aparticular chemical substrate. Thus, if the particular substrate flowsover, or otherwise encounters, the oxidizing enzyme bound to a targetanalyte and magnetic particle at an electrochemical cell, an oxidationreaction occurs. In such embodiments, electrons are accordingly releasedfrom a working electrode of the electrochemical cell to replenishelectrons stripped from the substrate by the oxidizing enzyme in aquantity proportional to the amount of target analyte present. Therelease or flow of electrons results in a current, which is detectableby an electrode, for example, as a change in current or a change involtage.

In other embodiments, such as the embodiment represented by theschematic diagrams of FIGS. 2A and 2B, the sample preparation reagentsat least include a population of magnetic particles 220, each having anaffinity molecule 230 bound to its surface. In some such embodiments, acompetitive binding agent 240 and a sample containing target analyte 210are added to the sample preparation reagents. The competitive bindingagent 240 of various embodiments includes a pre-bound target analyte270, which comes pre-bound to a signaling agent 250, for example, any ofthe signaling agents described above. The pre-bound target analyte 270may be indirectly bound to the signaling agent 250, for example, via anantibody, a nucleic acid probe, a nucleic acid aptamer, or otheraffinity molecule 260. In various embodiments, the unbound targetanalyte 210 from a sample and the competitive binding agent 240 competewith each other to bind to the affinity molecules 230 on the magneticparticles 220. The amount of competitive binding agent 240 and signalingagent 250 that successfully binds to the magnetic particles 220 isinversely proportional to the amount of unbound target analyte 210present in a sample. In embodiments where the signaling agent 250 of thecompetitive binding agent 240 is an oxidizing enzyme, an oxidationreaction occurs if a particular substrate flows over, or otherwiseencounters, the magnetic particles bound to the competitive bindingagents 240 at an electrochemical cell. Electrons are accordinglyreleased from a working electrode of the electrochemical cell toreplenish electrons stripped from the substrate by the oxidizing enzymein a quantity inversely proportional to the amount of target analytepresent in the sample. The release or flow of electrons results in acurrent, which is detectable by an electrode, for example, as a changein current or a change in voltage. In some embodiments, the use of sucha competitive binding technique advantageously allows for detection of atarget analyte even when the target analyte is present in a sample invery low concentrations.

In some embodiments contemplated herein, the sample reagents includeonly one population of magnetic particles and one population of detectoragents or competitive binding agents. Such embodiments are tailored fordetection of a single target analyte of interest.

In other embodiments, multiple populations of magnetic particles anddetector agents and/or competitive binding agents are provided, eachpopulation constructed to have its own affinity. In such embodiments,each population of magnetic particles has a unique affinity moleculebound to its surface, and each population of magnetic particles isthereby designed to bind with a different target analyte. Similarly,each population of detector agents includes a unique affinity moleculeand is thereby designed to bind with a different target analyte. Inembodiments employing the competitive binding approach, each populationof competitive binding agents includes a different pre-bound targetanalyte and is thereby designed to compete with a different targetanalyte. Such embodiments allow for the detection of a plurality oftarget analytes.

Those skilled in the art will appreciate that the possibilities forforming the magnetic particle-bound complexes are numerous and all suchpossibilities are contemplated herein. For example, the samplepreparation reagents may include a biotin-labelled antibody, which bindsto a portion of the target analyte. In some embodiments, antibodiesand/or nucleic acids present among the sample preparation reagents maybe pre-biotinylated such that a streptavidin conjugated signaling enzymecan bind with the biotinylated detector to form a complex. One suchstreptavidin conjugated signaling enzyme is HRP. The tagging combinationis not limited to biotin-streptavidin. Any suitable tagging scheme willwork. In another example, multiple HRP enzymes are conjugated togetherinto a molecule commonly known as a Poly-HRP molecule in order toenhance the signal generating capability of the resultant sandwichcomplex.

In addition to the components that form the magnetic particle-boundcomplexes, the sample preparation reagents of various embodiments caninclude one or more of: (a) agents that facilitate formation of themagnetic particle-bound complexes, such as salts; (b) agents thatfacilitate access and specificity to target analytes, such as detergentsand enzymes for lysis of bacteria or viruses or cutting of largemolecules or nucleotides; (c) blocker proteins to decrease nonspecificbinding; and (d) stabilizers such as, for example, trehalose, which canimprove the shelf life of the sample preparation reagents.

In at least some embodiments of the sample preparation reagents, saltsare necessary to enhance the likelihood of binding. For example, someembodiments include phosphate buffered saline (PBS). In otherembodiments, any salt which does not interfere with electrochemicaldetection may be provided within the reagents.

Blocker proteins, such as the well-known Bovine Serum Albumin, casein,fibrinogen, or other blocker protein may be provided to help stabilizethe antibodies, enzymes, and/or other proteins present among the samplepreparation reagents. Such blocker proteins may also help preventnon-specific binding of signaling enzymes to the magnetic particles andto the walls of the systems and devices described elsewhere herein.

Additionally, for embodiments that require lysis to access the moleculesor nucleic acids of interest, detergents may be employed. In variousembodiments, nonionic detergents, rather than ionic detergents, areprovided to prevent denaturation of the signaling enzyme and/orantibodies. Detergents can enhance lysis of bacteria, but are alsouseful for gently lysing various viruses, such as the influenza virus.Such lysing may be desirable to improve access to target analytes suchas nucleoproteins internal to a virus. Additionally, in someembodiments, the sample preparation reagents include enzymes thatenhance lysis and reduce viscosity during lysis; such reagents may benecessary for the preparation of some samples, for example, samplescontaining bacteria such as E. coli. The enzymes that enhance andfacilitate lysis may include lysozymes and DNAses that chop up releasedgenomic DNA without disrupting nucleic acid probes on the surface of themagnetic particles.

Enzymes such as RNAses or DNAses, which selectively chop largernucleotide sequences into smaller sequences, can be useful forgenerating smaller fragments having favorable binding kinetics. Suchenzymes are present in the sample preparation reagents of someembodiments. Other components may also be included within the samplepreparation reagents. For example, a stabilizer agent such as trehalose,may be present; such stabilizer agents help protect proteins fromoxidation and thereby increase the shelf-life of the reagents,especially at room temperature.

Those skilled in the art will appreciate that one or more of theabove-mentioned sample preparation reagents and chemical reactions maybe employed to test for any number of target analytes. Target analytesmay be detected and/or quantified in order to detect the presence ofabsence of a particular disease, infection, or health condition or tomonitor the progression of one or more metrics of health or disease.Suitable target analytes include, but are not limited to, one or more ofthe following: Vitamin D; cholesterol; insulin; C-Reactive Protein(CRP), which is an indicator of inflammation; free testosterone;cortisol, which is an indicator of stress; a biomarker associated withthe onset of infection; luteinizing hormone, which is an indicator ofovulation; follicular stimulating hormone, which is an indicator offertility; human chorionic gonadotropin (hCG), which is a biomarkerassociated with pregnancy; biomarkers for bacterial or viral infections,e.g., biomarkers for influenza A, B, and/or C, a biomarker for bird flu,a biomarker for swine flu, a biomarker for ebola, streptolysin O, whichis a biomarker for group A streptococcus, CD4+ and/or CD8 T-cells, whichserve as biomarkers for HIV/AIDS, and/or a biomarker for Bordetellapertussis; fetal DNA or DNA fragments, such as DNA fragments associatedwith genetic disorders; CA-153, which is a biomarker associated withovarian cancer; Prostate Specific Antigen (PSA), which is a biomarkerassociated with prostate cancer; and/or any other biomarker associatedwith a cancerous tumor, e.g., circulating tumor cells or othertumor-specific markers. In some embodiments, a plurality of targetanalytes may be detected and quantified to detect the presence orabsence of a particular disease, infection, or health condition or tomonitor the progression of health or disease. For example, in oneembodiment, leukocyte esterase, 16s ribosomal nucleic acids found inurinary tract-causing bacteria, and/or nitrites may be quantified todetermine the presence or absence of a urinary tract infection.

In some embodiments, the affinity molecule bound to the magneticparticle is an antibody that specifically recognizes and binds to thetarget analyte (referred to herein as “an antibody of the targetanalyte”). For example, when the target analyte is streptolysin O, theaffinity molecule is anti-streptolysin O (ASO).

In some embodiments, when a competitive binding agent is provided, thecompetitive binding agent is a compound comprised of the target analytepre-bound to a signaling agent. For example, in one embodiment where thetarget analyte is Vitamin D, the affinity molecule is a Vitamin Dprimary antibody, and the competitive binding agent is an HRP-conjugatedVitamin D molecule. In such an embodiment, the Vitamin D primaryantibody is immobilized on a surface of a magnetic particle, and anyVitamin D present within a sample competes with the HRP-conjugatedVitamin D molecules for binding with the antibody. In another example,the target analyte is testosterone, the affinity molecule is atestosterone primary antibody, and the competitive binding agent is anHRP-conjugated testosterone molecule. In such an embodiment, thetestosterone primary antibody is bound to a magnetic particle surface,and any testosterone present in a sample competes with theHRP-conjugated testosterone molecules for binding to the testosteroneprimary antibody.

In some embodiments, when a detector agent is provided, the detectoragent is a compound comprised of an antibody of the target analyte boundto a signaling agent. The antibody included within the detector agent isdifferent than the antibody that is immobilized on the magnetic particlesurface. In various embodiments, the two antibodies have specificity todifferent epitopes on the target analyte. As an example, in oneembodiment where the target analyte is CRP, the affinity molecule isCRP-specific antibody, and the detector agent is a different CRP-primaryantibody bound to a soybean peroxidase (SBP) molecule. The CRP-specificantibody bound to the magnetic particle has specificity to a differentepitope on the CRP than the CRP-primary antibody bound to the SBPmolecule. In such an embodiment, if the target analyte, CRP, is presentwithin a sample, one portion of the CRP binds to the affinity moleculeand another portion of the CRP binds to the detector agent, forming asandwich complex.

The target analytes may be detected and/or quantified using the systemsprovided herein. Various embodiments of the systems described herein aredesigned to create a self-contained environment in which any of thechemical reactions described above can occur in an automated mannerentirely or substantially without human intervention. For example, insome designs described herein, one or more of the above-describedchemical reactions proceeds without any need for an operator to add orremove reagents from the system. In certain embodiments, the systems areclosed such that biohazard risks, such as the risk of spilling samplecollected from a specimen, are minimized. In various embodiments, suchsystems include at least, a sample collection device, a cartridgedevice, and a reader device. Some exemplary embodiments of such devicesare described in detail below.

The Sample Collection Device

The sample collection device of various embodiments is configured tocollect a sample from a specimen. Sample collection devices may beconfigured to collect cells and other biological material from anydesired region or location, for example, an inner cheek, the mouth, thethroat, a nasal passageway, an ear, from urine, from saliva, from blood,or from another body part. One exemplary sample collection deviceincludes a unit that wicks a small droplet of blood, saliva, mucus, orurine into a small capillary channel. In other embodiments, the samplecollection device may be configured to collect biological material,particulates, or other chemicals from the environment, such as, forexample, from the air or the water, or from a physical surface or otherstructure.

The sample collection device of various embodiments is sized and shapedto collect a sufficiently large sample from an appropriate location of aspecimen such that it is possible, using the other devices describedbelow, to detect the presence, absence, and/or quantity of a targetanalyte in the specimen. For example, for some target analytes, such asones associated with the flu or cold viruses, the sample collectiondevice may be a nose-insertion swab; the swab is sized and shaped tocollect a sufficient amount of sample from a nasal passageway of anindividual to enable detection of target analytes associated with theflu or cold virus, if present in the individual. For other targetanalytes, such as, for example, ones associated with strep throat, thesample collection device may be a throat swab shaped to scrapesufficient cells from an individual's throat. As another example, thesample collection device appropriate for collecting a target analyteassociated with HIV may comprise a blood lancet. A blood lancet may alsobe provided in a system designed to detect and/or analyze levels ofcirculating analytes, e.g., Vitamin D, CRP, luteinizing hormone,follicular stimulating hormone, hCG, free testosterone, cortisol, virusand/or bacteria and/or cancer biomarkers, for example. In anotherexample, a sample collection device configured to collect saliva may beappropriate for collecting target analytes for various tests, including,for example, tests for tracking hormone levels (e.g., free testosteronelevels, cortisol levels, luteinizing hormone levels, or follicularstimulating hormone levels), drug levels, vitamin levels (e.g., VitaminD levels), pregnancy (e.g., hCG levels), and/or levels of biomarkersknown to be associated with particular viruses, bacteria, or cancer. Asample collection device configured to collect urine may be appropriate,for example, for collecting target analytes associated with pregnancy(e.g., hCG).

One such embodiment of a sample collection device is provided in FIGS.3A-3D. The sample collection device 300 is configured to collect a smallquantity of urine from a specimen. The sample collection device 300 hasa shaft 310, a collection head 320, a tip 330, and a collection area340, the collection area 340 formed of a capillary tube. The shaft ofsome embodiments is elongated to facilitate easy and sanitarycollection, with a collector's hand removed from the site of collection.The collection head 320 having a tip 330 is shown in isolation in FIGS.3C and 3D. In some embodiments, the collection head 320 is combined witha shaft having one or more of the features described in more detailbelow, such as, for example, complementary threading or a lockingmechanism and/or a sealing mechanism for engagement with a cartridgedevice.

Another embodiment of a sample collection device 400 is provided inFIGS. 4A and 4B. The provided sample collection device 400 is a nasalswab configured for collecting biological material from a nasal passage.The sample collection device 400 has a shaft 410, a collection head 420,and a tip 430. In some embodiments, the tip 430 is rounded; in otherembodiments, any blunt or substantially blunt tip shape may be used. Invarious embodiments, the shaft 410 is elongated to fit within the noseof an individual and the collection head 420 is configured to gentlyscrap against an inner wall of the nose to collect fluid, cells, and/orother biological material present within the nose. In some embodiments,the shaft 410 and the collection head 420 are formed of the samematerial; in other embodiments, they are formed of different materials.In some embodiments, both the shaft 410 and the collection head 420 areformed of a plastic. In some embodiments, the sample collection device400 is pre-packaged within sterile packaging and is configured forone-time use.

In some embodiments, the tip 430 of the sample collection device 400 isblunt and includes no sharp edges; the blunt design reduces the risk ofusers hurting themselves on the sample collection device. Additionally,advantages of a blunt tip 430 are explained in more detail below in thediscussion of the cartridge device. The sample collection device 400 ofvarious embodiments is configured for full or partial insertion intosuch a cartridge device.

In various embodiments of the sample collection device, including samplecollection device 400 of FIGS. 4A and 4B, the device includes aplurality of functional components. Such functional components arerepresented schematically in the block diagram of FIG. 5. As thesecomponents are described functionally, one skilled in the art willappreciate that the components may take many physical forms. Allsuitable physical forms are herein contemplated and incorporated. Asdepicted, in various embodiments, the sample collection device 500includes one or more of: a collection zone 510 for collecting the sampleand storing the sample for delivery to a reservoir within a cartridgedevice; a sealing zone 520 for facilitating the formation of aliquid-tight seal between the sample collection device 500 and acartridge device upon insertion of the sample collection device 500 intothe cartridge device, a locking zone 530 for facilitating a fixedengagement between the sample collection device 500 and the cartridgedevice such that upon insertion of the sample collection device 500 intothe cartridge device, the collection device is mated irreversibly andimmovably with the cartridge; and a handle zone 540 for the user tograsp and manipulate the sample collection device. In some embodiments,the collection zone 510 is also provided and configured to break amembrane within the cartridge device in order to obtain access into areservoir within the cartridge device. In some embodiments, the handlezone 540 is breakable or otherwise removable from the remainder of thesample collection device 500 following insertion of said remainder ofthe sample collection device 500 into the cartridge device.

One embodiment of a sample collection device 600 with the functionalzones prominently displayed is provided in FIG. 6. As shown, the samplecollection device 600 includes a handle 640 for holding the device 600,a locking feature 630 for locking the device 600 into a cartridge, asealing feature 620 for forming a liquid-tight seal with an internaltunnel in the cartridge, and a collection feature 610 for collecting andtemporarily storing a sample.

The Cartridge Device

In various embodiments, a cartridge is formed of a housing, whichdefines an enclosed space and has various features that enable thecartridge to do one or more of the following: receive a sample withtarget analytes from a sample collection device, store the sample withsample preparation reagents, provide a space for mixing and hybridizingthe target analytes with sample preparation reagents, provide ananalysis zone wherein hybridized target analytes localize over sensorsfor detection, provide a liquid medium for transporting the hybridizedtarget analytes to the analysis zone, store and provide a substrate thatcan undergo a detectable reaction when introduced to the hybridizedtarget analytes, provide a liquid medium for transporting the substrateto the hybridized target analytes in the analysis zone, and provide awaste collection zone where waste is stored.

In various embodiments, the cartridge is a substantially closed systemin which occur the reactions needed to detect the presence, absence,and/or quantity of target analytes. The cartridge of such embodiments issaid to be “substantially closed” because the only inputs needed intothe cartridge system are one or more of the following: a sample from aspecimen; energy to facilitate mixing, hybridization, and/or valveopening; and a magnetic force to facilitate localization of hybridizedtarget analytes within an analysis zone; the only outputs from thecartridge are electrical signals. In various embodiments, the cartridgeis target-analyte-specific with the included sample preparation reagentsselected to detect one or more specific target analytes. Differentcartridge types include different reagents intended to identifydifferent target analytes.

One embodiment of a cartridge 700 is provided in FIGS. 7A and 7B.Specifically, FIG. 7A depicts various non-limiting examples ofcomponents of a cartridge 700 coupled together in a fixed configuration;FIG. 7B depicts the same components separated, prior to assembly, inorder to highlight various features of the cartridge 700. As shown, thecartridge 700 of various embodiments includes a housing 710 formed of acover component 720, an internal component 730, and a base component740. Upon assembly, these components are coupled together to form afixed structure having an input tunnel 712, a plurality of reservoirs722, and an analysis channel 732. In some embodiments, these componentsare formed of a hard plastic or other substantially rigid material.

The various components of a similar cartridge embodiment and theorientation of the components relative to each other are also shown inthe exploded view of FIG. 8. As shown, upon assembly of the depictedembodiment, the cover component 820 is disposed on a first side of theinternal component 830, and the base component 840 is disposed on asecond side of the internal component 830. A circuit board component 850is positioned between the internal component 830 and the base component840 and attached to the internal component 830, for example, with alayer of adhesive 860. Features of the cover component 820 and the firstside of the internal component 830 together define an input tunnel 812and a plurality of reservoirs 822, and features of the second side ofthe internal component 830 and the circuit board 850 define an analysischannel 832.

The various components of another cartridge embodiment and the assemblyof such components are shown in the exploded, semi-exploded, andnon-exploded perspective views of FIGS. 9A-9C, respectively. As shown,during assembly of the cartridge 900, the first cover component 920 isdisposed laterally of the internal component 930, and the second covercomponent 940 is disposed on the opposite lateral side of the internalcomponent 930. A circuit board component 950 is attached to the internalcomponent 930, for example, to an underside of the internal component930 using a layer of adhesive. In such embodiments, the internalcomponent 930 and circuit board component 950 are positioned togetherbetween the first cover component 920 and the second cover component940. Features of one or more of the first cover component 920, thesecond cover component 940, and the internal component 930 may togetherdefine an input tunnel 912, and features of the underside of theinternal component 930 and the circuit board 950 may define an analysischannel 932. In some embodiments, the internal component 930 defines aplurality of reservoirs. In some such embodiments, each reservoir is awell that has been etched, carved, cut, or otherwise formed into areservoir-defining portion 922 of the internal component 930. In someembodiments, the open side of each reservoir is covered by agas-permeable/liquid-impermeable membrane.

Returning to the cartridge embodiment 800 of FIG. 8, various elements ofthe internal component 830 are also shown in the top view and partialperspective view of FIGS. 10A and 10B. In the depicted views, the inputtunnel 812 leads to a first reservoir 824 in the cartridge 800. A secondreservoir 828 and third reservoir 826 are also provided with the firstreservoir 824. Each of the plurality of reservoirs 824, 826, 828 has acorresponding outlet near a bottom portion of the reservoir, which opensto the microfluidic analysis channel 832.

One skilled in the art will appreciate that while three reservoirs aredepicted, in various embodiments, the plurality of reservoirs mayinclude two reservoirs or four or more reservoirs and may adoptalternative spatial configurations. Any and all possible spatialconfigurations are contemplated and expressly incorporated herein. Anexample of another possible spatial configuration is provided in FIGS.11A-11C. FIGS. 11A-11C depict the internal component 1130 and thecircuit board component 1150 of a cartridge embodiment with the externalhousing components removed. In the depicted embodiment, the reservoirs1122 are oriented in a cloverleaf fashion around an analysis channel1132. As in other embodiments, the input tunnel 1112 extends from anaperture 1102 of the cartridge to a first reservoir 1124, and theanalysis channel 1132 is defined by walls of the internal component 1130and a wall of the circuit board component 1150. Additionally, eachreservoir 1122 includes an outlet 1123, which connects the reservoir1122 to the analysis channel 1132, and the analysis channel 1132 extendsfrom the reservoirs 1122 to an absorbent pad 1136. In the depictedembodiment, sensors 1158 on the circuit board component 1150 arepositioned within the analysis channel 1132. Additionally, in thedepicted embodiment, a sonicator element 1121 is included, the sonicatorelement 1121 positioned to form all or a portion of the bottom surfaceof the first reservoir 1124.

In various embodiments of the cartridge device and sample collectiondevice, such as, for example, in all embodiments described above, theinput tunnel of the cartridge is configured to receive all or a portionof the sample collection device. One example is provided in FIGS. 11Aand 11B, using the cartridge 800 of FIG. 8 and the sample collectiondevice 400 of FIGS. 4A and 4B. As shown, the input tunnel 812 of thecartridge 800 is sized and shaped to receive all or a portion of thesample collection device 400. In certain embodiments, the input of acollected sample occurs by advancing all or a portion of the samplecollection device 400 into the cartridge 800. For example, in FIGS. 11Aand 11B, the sample collection device 400 is slid, tip 430 first, intothe input tunnel 822. The sample collection device 400 is slid into theinput tunnel 822 until all or a portion of the head 420 of the samplecollection device 400 is disposed within the first reservoir 824.

In some embodiments, prior to insertion of the sample collection device400 into the cartridge 800, an internal membrane is disposed within theinput tunnel or between the input tunnel and the first reservoir. Oneembodiment of an internal membrane 823 is visible in FIG. 10A. While theinternal membrane is most visible in FIG. 10A, it is contemplated thatany and all of the cartridge embodiments provided herein may alsoinclude an internal membrane. As depicted, the internal membrane 823covers, at least, the entirety of the cross-sectional area of the inputtunnel 812, at or near the entryway to the first reservoir 824. Theinternal membrane 823 of some embodiments is double-walled and containsa volume of liquid between the two walls. The membrane liquidfacilitates suspension of the sample from the sample collection device400 and helps transport the sample particles into the first reservoir824. In embodiments employing the competitive agent detection methoddescribed above, the internal membrane 823 also stores the competitivebinding agents. In various embodiments, any or all of the samplepreparation reagents, including for example, the magnetic particles,competitive binding agents, and detector agents, may be stored on orwithin the internal membrane 823.

In various embodiments, insertion of the sample collection device 400into the input tunnel 812 ruptures the internal membrane 823, therebyreleasing any stored liquid, any stored reagents, and the collectedsample particles into the first reservoir 824. In other embodiments, asdescribed below with reference to FIGS. 13A and 13B, the internalmembrane 823 of cartridge 800 is a thin, single-walled membrane. In somesuch embodiments, one or more of the above-mentioned molecules areprovided dry-stored on the membrane. As used herein, “dry-stored on themembrane,” means the molecules have been attached covalently,non-covalently, or electrostatically, or otherwise adhered to a drysurface of the membrane. In a thin, single-walled membrane, the drysurface is the surface facing the input tunnel.

Another configuration for the internal membrane is provided in FIGS. 13Aand 13B. FIGS. 13A and 13B schematically represent a top view and aperspective view of a first reservoir 1324 (similar to first reservoir724 or 824) shown in isolation, removed from the remainder of thecartridge in order to highlight the placement of the internal membrane1323. In the depicted embodiment, the internal membrane 1323 is disposedon an outer wall of the first reservoir 1324. Such a membrane 1323 wouldbe within the input tunnel or within a space between the input tunneland the first reservoir 1324. The internal membrane 1323 blocks entry toa sample input aperture 1321, thereby preventing liquid stored withinthe first reservoir 1324 from leaking out of the reservoir into, forexample, the input tunnel. In some such embodiments of the internalmembrane 1323, various molecules 1319, such as, for example, competitivebinding agents, signaling agents as depicted in FIGS. 1A-1D as 150, orany other sample preparation reagents may be attached to a dry surfaceof the internal membrane 1323.

In various embodiments of the input tunnel, the tunnel includes aplurality of functional components that are complementary to functionalzones and features of the sample collection device of variousembodiments. Such functional components are represented schematically inthe block diagram of FIG. 14. As these components are describedfunctionally, one skilled in the art will appreciate that the componentsmay take many physical forms. All suitable physical forms are hereincontemplated and incorporated. As depicted, in various embodiments, theinput tunnel 1400 includes one or more of: an entry port zone 1410 thatprovides an opening through which the sample collection device can enterthe tunnel; a guidance zone 1420 for directing the collection devicealong an axis towards a first reservoir and restricting movement that isnot along the axis; a locking zone 1430 with mechanical features tocomplement the locking zone on the sample collection device forachieving a secure, fixed coupling between the two devices; a sealingzone 1440 with mechanical features to complement the sealing zone on thesample collection device for achieving a liquid-tight seal between thetwo structures; and a membrane zone 1450 wherein a membrane is affixedto prevent leakage from the first reservoir. The first reservoir 1460 isalso provided as it may form the distal end of the input tunnel 1400.

One embodiment of an input tunnel 1500 with the functional zonesprominently displayed is provided in FIGS. 15A-15C. As shown, the inputtunnel 1500 is defined, at least in part, by the internal component1501. The input tunnel 1500 includes: an aperture 1510 through which thesample collection device can enter the tunnel 1500; an elongated portion1520 for directing the collection device along an axis towards a firstreservoir, the elongated portion 1520 having a diameter which restrictslateral movement of the sample collection device; a locking zone 1530with mechanical features to complement and fixedly couple the samplecollection device; a sealing zone 1540 with a narrowed diameter, agasket, and/or other mechanical feature to help achieve a liquid-tightseal between the internal tunnel 1500 and the sample collection device;and a membrane 1550. Also visible are a plurality of reservoirs 1560. Avent 1570 is also provided within the input tunnel 1500 to allow for thedisplacement of air that may otherwise create a pressure resisting theinput of the collection device into the tunnel 1500.

As mentioned above, various embodiments of the cartridge include amembrane that prevents liquid from flowing out of the first reservoirand into the input tunnel prior to insertion of a sample collectiondevice. In such embodiments, the sample collection device ruptures theinternal membrane while advancing into the first reservoir. In certainembodiments, two events happen at, or substantially at, the instant thesample collection device pushes the membrane to its rupture point: (1) aflexible feature, such as for example, a rubber gasket or a gasket ofany other suitable material, at the base of the collection head movesinto position to form a liquid-tight seal with the structural housingfeatures surrounding the membrane, and (2) the shaft of the samplecollection device advances to a location where it locks in place withinthe input tunnel of the cartridge. The locking may be achieved, forexample, by providing complementary grooves and ridges, grooves andteeth, or other complementary features between the shaft of the samplecollection device and the surrounding input tunnel. By entering into astructurally engaged, fixed configuration within the input tunnel, thesample collection device of various embodiments is able to remain inplace and resist pressure exerted on the collection head during ruptureof the membrane. Additionally or alternatively, such a configurationenhances the convenient disposal of the cartridge after use bypreventing users from accidentally opening the cartridge, therebypreventing exposure to the cartridge's potentially bio-hazardouscomponents.

FIGS. 16A-16C depict one example of a sample collection device in alocked engagement within the input tunnel embodiment of FIGS. 15A-15C.In the depicted example, the sample collection device is the samplecollection device 600 from FIG. 6. As shown in FIG. 16B, in the lockedposition, complementary features 630, 1530 on the shaft of the samplecollection device 600 and the surrounding input tunnel 1500 engage, andas shown in FIG. 16C, in the locked position, the membrane 1550 hasruptured and a sealing mechanism 1540 on the collection device 1500 hasformed a seal with a sealing portion 620 of the input tunnel 600. In thedepicted embodiment, the sealing portion 1540 of the input tunnel 1500includes a tunnel portion having a narrowed diameter and the sealingportion 620 of the collection device includes a gasket.

Returning to FIGS. 12A and 12B as another example, during insertion ofthe sample collection device 400 into the cartridge 800, the samplecollection device 400 ruptures the internal membrane 823 while advancinginto the first reservoir 824. In various embodiments, the tip 430 of thesample collection device 400 is blunt to ensure the internal membrane823 deforms then ruptures at a controlled rupture point rather thanbeing immediately pierced by the tip 430.

In order to obtain an internal membrane, such as, for example, internalmembrane 823, having the desired rupture characteristics and desiredrupture point, in various embodiments, the internal membrane is formedof a material carefully selected to have a desired modulus ofelasticity, yield point, and/or rupture point. The modulus of elasticityis a constant that characterizes a material's degree of elasticity andcan be used to determine the maximum the membrane can be stretched whilestill returning to its original shape. This point is called the yieldpoint. Beyond the yield point, the material exhibits plasticity,undergoing irreversible deformation. Beyond the yield point is anothercritical point called the rupture point. The rupture point is when themembrane fails or breaks. The specific modulus of elasticity desired foran embodiment varies according to the size and shape of the samplecollection device tip, which exerts pressure onto the internal membrane.The selected membrane material may include, for example: polyurethane,polysilicone and polybutadiene, nitrile, or other elastic material orcomposite thereof. Other suitable materials for the deformable membraneinclude, for example, parafilm, latex, foil, and polyethyleneterephthalate.

In various embodiments, the size of the collection head 420, the shapeof the tip 430, the rupture point of the internal membrane material, andthe location of the complementary locking features are selected inconsideration of each other.

In one embodiment, the complementary locking features include positivegrooves (i.e., ridges or other protrusions) radially placed in the inputtunnel and negative grooves or other complementary depressions radiallyplaced on the shaft of the sample collection device. The radialplacement allows for insertion of the sample collection device 400 intothe input tunnel 812 regardless of the radial orientation of the samplecollection device 400. In other embodiments, one or a plurality ofnon-radial complementary engagement features may be provided. In someembodiments, the engagement features are constructed such that, when theengagement features of the shaft 410 move against the engagementfeatures of the input tunnel 812, one or both of the engagement featuresare reversibly compressed or retracted, returning to their initialpositions when the shaft 410 enters the location of fixed engagement.Such a structure prevents any further forward or backward lateralmovement of the sample collection device 400. Such a structure providestactile confirmation for the user that the sample collection device wasinserted fully and correctly; additionally, the two-way lock givesstructural support to the rupture/seal mechanism. By preventingintentional and accidental removals of the sample collection device 400from the cartridge 800, the risk of contact with the sample isminimized. Accordingly, the biohazard risk is minimized. Such astructure allows for easy disposal of the system into the normal trash.

Within the cartridge 800, the input tunnel 812 of some embodimentsextends from an aperture on a surface of the cartridge 800 to a firstreservoir 824. In the depicted embodiment, the plurality of reservoirsincludes a first reservoir 824, a second reservoir 828, and a thirdreservoir 826. In other embodiments, only two or four or more reservoirsmay be present. These reservoirs 822 are each separate from the othersand no cross-mixing of their contents occurs within the reservoirs. Asvisible in the top and perspective views of FIGS. 10A and 10B, each ofthe plurality of reservoirs 822 is, at least at times, in fluidconnection with a microfluidic analysis channel 832 by way of areservoir outlet. In certain embodiments, the bottom “floor” or bottominternal surface of each reservoir is not flat, but rather, angleddownward toward the outlet, with the intersection of the reservoir andthe analysis channel 832 located at the lowest height or deepest depth.Such embodiments help encourage flow of all reservoir contents into theanalysis channel 832, thereby minimizing dead volume. In variousembodiments, each reservoir outlet has a valve disposed therein (suchas, for example, valves 825, 827, 829), which fully seals the outlet andprevents liquid from flowing from the reservoirs into the analysischannel 832 prior to use. In use, in accordance with a method describedin more detail below, the plurality of valves can open in a timed mannersuch that contents from each of the plurality of reservoirs 822 can flowsequentially into the analysis channel 832.

In the depicted embodiment, the first reservoir 824 is furthestdownstream and closest to the input channel 822. This is by design sothat, upon insertion of the sample collection device 400, the head 420enters the first reservoir. Upon insertion of the sample collectiondevice 400, the first reservoir 824 is at least partially filled withthe sample preparation reagents described above and a first liquid.Within this disclosure, the terms “first reservoir” and “samplepreparation reservoir” may be used interchangeably. In some embodiments,the sample preparation reagents and a first liquid are stored in thefirst reservoir 824 prior to use; in other embodiments, one or more ofthe sample preparation reagents are stored on a membrane between theinput tunnel 812 and the first reservoir 824 prior to use. In variousembodiments, when the sample collection device 400 enters the firstreservoir 824, the first reservoir 824 also becomes filled with sampleparticles, including one or more target analytes, if present in thesample. Additionally, in various embodiments, when the sample collectiondevice 400 enters the first reservoir 824, the liquid is gently mixed tosuspend and hybridize particles within the reservoir. In someembodiments, the target analytes in the sample hybridize and bind, atleast, to the magnetic particles and affinity molecules present amongthe sample preparation reagents, forming magnetic particle-boundcomplexes. When the first valve opens, liquid from the first reservoir824 acts as a transport medium causing the magnetic particle-boundcomplexes and other particles to flow from the first reservoir 824 intothe analysis channel 832. Advantageously, the liquid serving as themixing medium and storage medium within the first reservoir 824 alsoacts as the flow medium to transport the contents of the first reservoir824 to an analysis zone within the analysis channel 832 without the needfor a pump.

The second reservoir 828, present in some but not all embodiments, is atleast partially filled with a wash solution. The term “second” as usedherein, refers to the order in which solution from the reservoir isreleased into the analysis channel 832 rather than the position of thereservoir within the cartridge 800. The second reservoir 828 is locatedfurthest upstream in various embodiments. In such embodiments, when acorresponding second valve 829 opens, the wash solution flows from thesecond reservoir 828 into the analysis channel 832, thereby removing allor substantially all unbound detector agents and/or unbound competitivebinding agents from the analysis channel 832. Locating the wash solutionin the upstream-most reservoir ensures that all free-floating, unboundmolecules from the sample preparation reservoir 824 are washed from theanalysis channel 832 and reduces the likelihood of having anynon-specific binding of significance occur within an analysis zone ofthe analysis channel 832.

The third reservoir 826 is located upstream of the first reservoir 824,for example, between the first reservoir 824 and the second reservoir828. The third reservoir 826 is at least partially filled with achemical substrate in solution. In various embodiments, the solution ofthe third reservoir 826 includes a substrate that undergoes a reactionin the presence of a signaling agent from the first reservoir 824. Forexample, in some embodiments, the substrate of the third reservoir 826undergoes an oxidation reaction in the presence of an oxidizing enzymefrom the first reservoir 824. In various embodiments, when the thirdvalve 827 opens, liquid from the third reservoir 826 acts as a transportmedium causing the chemical substrate to flow from the third reservoir826 into the analysis channel 832.

In various embodiments, liquid flows from each of the plurality ofreservoirs 822 into the analysis channel 832 and continues to flow in adownstream direction within the analysis channel as a result ofcapillary action. In certain embodiments, a vent is provided in or overeach reservoir to allow for air to replace the liquid emptying from eachreservoir into the analysis channel. Without proper ventilation, fluidmay not flow within the cartridge. In some embodiments, the vent isformed by placing an air permeable membrane, such as, for example, aPTFE membrane, over the plurality of reservoirs. In some suchembodiments, at least portions of the cover component of the cartridgehousing may be formed of PTFE; in other embodiments, an opening may beprovided in the cover component over the reservoirs, which is sealedwith a PTFE membrane. Advantageously, a membrane such as a PTFE membranethat is air permeable but not liquid permeable provides a means forsealing off the top of each reservoir to prevent liquid leakage whileallowing for the liquid to drain out of the reservoir into the analysischannel. Additionally, one or more vents 835, 836 may be provided overall or a portion of the analysis channel, in order to allow displacedair to vent as the liquid flows into the channel. Bubbles are often aproblem in microfluidic systems. This issue is countered in someembodiments with the strategic placement of the vents, which allow forpassive degassing of bubbles. For example, in some embodiments, all or aportion of the top side of the microfluidic channel (within the internalcomponent of the cartridge) is replaced with a PTFE membrane or otherair permeable membrane. In such embodiments, the membrane forms theceiling of much of the channel. The pore sizes of the membrane can varyand can be selected to include pores of 0.1 microns to 3 microns indiameter. In some such embodiments, the membrane is sealed onto thechannel and/or over the reservoirs with adhesive.

Attachment of an air-permeable membrane within the cartridge duringassembly may be achieved using any suitable manufacturing process. Insome embodiments, adhesive is applied to a bottom side of the membrane,and the membrane is taped to a bottom wall of the analysis channel; thebottom wall of the channel is formed of a surface of the circuit boardcomponent. A vacuum is then applied by pushing air through one or morevents; the vacuum raises the membrane such that an adhesive portion ofthe membrane contacts the side walls of the analysis channel, forming anadhesive seal. In effect, the membrane will be sucked into place andbonded to the side walls of the analysis channel through the use of anapplied vacuum and adhesive.

To facilitate capillary flow in the analysis channel, in variousembodiments, the interior surfaces of the channel are made to behydrophilic. As used herein, “hydrophilic” refers to an affinity for asurface and/or molecule to maximize its contact area with water. Ahydrophilic surface is one in which the contact angle of a droplet ofwater is less than 90 degrees. In some embodiments described herein,surfaces having a contact area of less than 60 degrees are achieved. Asused herein, “capillary flow” or “capillary action” refers to movementof fluid along a fluidic channel driven by at least two physicalproperties of the fluid and the channel. The physical propertiesinclude: hydrophilic adhesion of the molecules of the fluid in contactwith surfaces of the channel, and intermolecular cohesive forces withinthe body of liquid which help to draw the bulk of the fluid along as themolecules closest to the hydrophilic surfaces of the channel propagatealong the channel surface.

In various embodiments, the analysis channel is defined by two or morewalls, and some or all such surfaces are made to be hydrophilic. In someembodiments, the analysis channel includes a first semi-circular wallformed into the internal component of the cartridge and a second wallformed of a surface of the circuit board component of the cartridge. Inother embodiments, such as, for example, the embodiment depicted by thecross-sectional view of an analysis channel in FIG. 17A, the walls ofthe analysis channel 1732 include three walls that are carved, etched,or otherwise formed into the internal component 1730 of the cartridgeand the fourth wall is formed of a surface of the circuit boardcomponent 1750.

Various materials or surface chemistry modifications can be used tocreate an analysis channel having hydrophilic walls. For example, theinternal component 1730 and the analysis channel walls formed of theinternal component 1730 may be made from a thermoplastic resin as shownin FIG. 17A. Such an embodiment is also depicted in FIG. 17B; in FIG.17B, an adhesive layer 1760 is also shown coupling the internalcomponent 1730 to the circuit board component 1750 to form the analysischannel 1732. As another example, such as, for example, the embodimentprovided in FIG. 17C, one or more surfaces of the internal component1730, including the surfaces forming walls of the analysis channel 1732,may undergo pegylation grafting mediated by plasma treatment to activatethe surfaces such that polyethylene glycol (PEG) will bond thereto,making a hydrophilic and protein-resistant modified surface 1731.Additionally, in some embodiments, a commercially available lateral flowtype membrane, may be disposed within the channel interior to provide awicking material within the channel.

As described above, in some embodiments, the cartridge includes a meansfor venting gases from the analysis channel. As shown in FIGS. 17D and17E, in some embodiments, the means for venting gases includes one ormore vents 1736, which are formed of small holes within the internalcomponent 1730. In some embodiments, the walls defining the vents 1736are hydrophobic and the holes are sufficiently small such that aqueousliquids within the analysis channel 1732 are repelled from the vents1736 and do not leak. As shown in FIG. 17F, in another embodiment, abubble bypass segment 1733, defined by the internal component 1730 isprovided in a top portion of the analysis channel 1732. The bubblebypass segment 1733 is sized and positioned to allow gases to flowthrough bubble bypass segment 1733 while liquids within the analysischannel remain within the lower, main segment of the channel 1732. Insome embodiments, the bubble bypass segments 1733 are provided betweentwo vents 1736 and serve to transport gases from the analysis channel tothe vents 1736 for release.

In other embodiments, the means for venting gases from the analysischannel 1732 includes a breathable membrane, such as a PTFE membrane,which replaces one analysis channel wall otherwise formed of theinternal component 1730. One such embodiment is depicted in FIG. 17Gwith a top wall of the analysis channel 1732 replaced by a breathablemembrane 1735. In some embodiments having a breathable membrane 1735 forventing, prewetting of the membrane 1735 may be required, because somebreathable materials, such as PTFE, are hydrophobic. To eliminate theneed for a distinct prewetting step, structural prewetting may beutilized in some embodiments. One such embodiments is depicted in FIG.17H. As shown, to “structurally prewet” a breathable membrane 1735,rails 1737 of hydrophilic material may be provided, which run the lengthof the breathable membrane. Such rails 1737 promote the flow of liquidalong the rails, for example, from a reservoir into the analysis channel1732 and/or along the length of the analysis channel 1732. Thehydrophilic rails 1737 help overcome the hydrophobic resistance of themembrane. These rails 1737 can be formed in a multitude of ways and areconstructed to include thin plastic rails spanning the length of themembrane ceiling 1735. In some embodiments, adhesives disposed directlyon the membrane can form the rail; in another embodiment, the rail maybe formed by a patterned surface modification of the membrane whichcauses a hydrophilic surface modification to run the length of theanalysis channel 1732.

Additionally, as described in more detail below, in some embodiments,one or more sensors are disposed on the circuit board component 1750within the analysis channel 1732. As depicted in FIGS. 17D-17I andspecifically identified in FIG. 17I, the sensor 1758 may be formed ofgold or other conducting metal, and as described below with reference toFIG. 18A, may include additional surface chemistry modifications 1757.In various cartridge embodiments described herein, such as, for example,in cartridge 700 of FIGS. 7A and 7B and cartridge 800 of FIG. 8, it iscontemplated that the analysis channel 732/832 may include any or all ofthe features described and/or depicted within any of FIGS. 17A-17I orany other features known to those skilled in the art.

Additionally, to facilitate flow via capillary action in the analysischannel, in various embodiments, an absorbent material is provided atthe downstream-most end of the analysis channel. One example of anabsorbent material, in the form of an absorbent pad 834, is visible inFIG. 10A. The absorbent material or pad 834 wicks liquid from theanalysis channel 832, thereby encouraging liquid to flow downstream tothe absorbent pad 834. In some embodiments, the absorbent pad 834 actsas a waste receptacle, collecting all waste liquids and waste particlesafter they have flowed through the analysis channel 832. In variousembodiments, the absorbent pad's size and degree of absorbency isselected to meter the flow of liquids and particles within the analysischannel 832. For example, in some embodiments, the volume of liquid thatthe absorbent pad 834 can wick must be great enough to drain all liquidfrom the first (sample preparation) reservoir 824 and the second (wash)reservoir 828 and draw the liquid carrying the chemical substrate fromthe third (substrate) reservoir 826. Such a condition may serve as thelower limit of absorbency. Additionally, acting as an upper limit is therequirement that the flow of the liquid carrying the chemical substratemust slow or stop over an analysis zone of the analysis channel 832 sothat the chemical substrate has time to react with signaling agentslocalized within the analysis zone.

As shown clearly, for example, in FIGS. 7B, 8, and 11A-11C, thecartridge of various embodiments also includes a printed circuit board,750, 850, and 1150, respectively, referred to herein as a circuit boardor circuit board component. The circuit board component is coupled tothe internal component of the cartridge. The circuit board component 750of FIG. 7B is provided, in isolation, in FIGS. 18A and 18B. The circuitboard component 750 includes electrical components, for example, one ormore of: a resistor, electrical leads 754, vias 756, and sensors 758needed for detection of target analytes. Although described separately,it is to be appreciated that electrical components of the circuit boardcomponent 750 need not be separate structural elements. One or moreelectrical components and/or circuits may perform some of or all theroles of the various components described herein. In some embodiments,the resistor is provided as a unique-identifying tag, which allows for areader device (described in more detail below) to distinguish betweencartridge types. The resistor may include a small surface mountresistor, a resistive-ink based resistive element, or any otherresistive element that allows the reader to “read” the resistor andthereby identify the cartridge type. As used herein, cartridges differin “cartridge type” if they are configured for the detection ofdifferent target analytes. In other embodiments, different non-resistivemeans of identifying the cartridge type are employed.

As described in more detail below, the electrical leads 754 of variousembodiments are provided to establish electrical connections andcontinuity with a reader device. As shown in FIG. 18B, the electricalleads 754 are electrically coupled to the vias 756, providing electricalcurrent to such components when activated by the reader device. A via isa standard product on printed circuit boards and is typically used toenable signal traces on one layer of a circuit board to continueelectrically with another layer. The vias provide electrical continuitythrough multiple layers. Such vias are excellent conductors of heat;they are able to transfer heat to a very precise location withoutaffecting the surrounding areas, because the surrounding material thatcomprises most circuit boards is an excellent insulator of heat. Thus,in various embodiments, a plurality of vias 756 are provided in thecircuit board component, and each via 756 is disposed under, over, oradjacent to a phase-changeable, heat-actuated valve disposed in areservoir outlet to create a valve actuating element. Together, the via756 and the valve form a valve unit. The precision of heat transferassociated with the vias 756 allows for minimal crosstalk between valveslocated close to each other; thus, the timing of valve actuation can becarefully controlled for each valve. In some embodiments, the valves areformed of a wax, for example, a hydrophilic wax, and the vias 756 act asconductors of heat to melt wax at precise points of time, as controlledby a reader device. One or more heating elements generate the heat thatis to be conducted to the exact location where the wax needs to bemelted. Upon melting of a wax valve disposed in the outlet of areservoir, the outlet is no longer occluded and the reservoir has anopening through which its fluid contents can drain into the analysischannel. The heating element of some embodiments forms part of thecircuit board component. For example, in the embodiment of FIG. 18B, theheating element is a resistive heating element appearing as a serpentinetrace 755 located on the bottom side of the circuit board component 750,surrounding the via 756. In other embodiments, the heating element islocated external to the cartridge, for example, on the reader. Invarious embodiments in which a resistive heating element is used, inorder to generate heat, current is allowed to flow through the resistiveheating element, for example, through actuation of a transistor. Currentpassing through the resistive heating element generates heat throughJoule heating. The heat is conducted to the via due to physical contactbetween the resistive heating element and the via. In variousembodiments, the heat is then conducted through the via up the waxbarrier and a phase transition, such as, for example, melting, of thewax occurs.

In order to ensure full melting of the wax with precise timing, invarious embodiments, the wax valves are carefully constructed within theoutlets of the reservoirs. For example, in some embodiments, it ispreferable for the wax valves to have the minimum height necessary toocclude the outlet of the reservoir; the minimal height minimizes thedistance heat must travel to melt the wax. One example method forrealizing a wax barrier having such characteristics involves applyingmelted wax to a pre-heated via. Advantageously, when the via ispre-heated, it takes longer for the wax valve to solidify relative to aroom-temperature via; thus the wax has more time to flatten and expandoutward before hardening. “Pancaking” of the wax is desirable tominimize the height, which will maximize the chance of proper meltingactuation of the valve. Additionally, the heating of the via facilitatesa greater level of contact area between the wax and the via such that agreater proportion of the wax experiences the heat, also maximizing thechance of proper valve actuation. The method of heating the via prior todeposition of wax is further enhanced with the following method: theopening of the reservoir is aligned over the via such that when themelted wax is applied to the pre-heated via, the opening at the bottomof the reservoir is spatially close to the via such that when the waxhardens, the wax adheres simultaneously to multiple inner walls of thereservoir and the via itself. This is advantageous for enhancing themanufacturing yield of intact valves that fully occlude the opening tothe analysis channel such that no inadvertent flow of liquid from thereservoir occurs.

A cross-sectional view of one embodiment of the valve 825 is provided inFIG. 19. The valve 825 is located within an outlet at the bottom of thereservoir 824 of the cartridge 800. As depicted in FIG. 19, thereservoir 824 is defined by walls of the internal component 830. In someembodiments, the outlet is formed of a hole within a bottom wall of theinternal component 830. In various embodiments, the circuit boardcomponent 850 is disposed below the internal component 830 and affixedto the internal component 830 with the use of an adhesive 860, such as,for example, a double-sided adhesive tape which may be hydrophilic tosupport the capillary flow of fluid. In various embodiments, the valve825 is formed of a heat-sensitive, phase-changeable material, such as,for example, a hydrophilic wax. Prior to actuation, the wax or otherheat-sensitive material of the valve 825 is in a solid or semi-solidstate and is sized and shaped to fill an entire cross-section of theoutlet such that no liquid can escape from the reservoir 824 into theanalysis channel 832. As depicted, the heat-actuated valve 825 of someembodiments is aligned directly above a via 856 or other localizedheat-conductive element. Such alignment allows for the localizedapplication of heat to induce a phase change in the valve 825 withoutcausing a phase change of any neighboring valves. In variousembodiments, the phase change melts or otherwise transforms theheat-sensitive material such that it no longer causes full occlusion ofthe outlet, but instead permits liquid in the reservoir 824 to flow intothe analysis channel 832.

In some embodiments, the wax material disposed upon the via, and whichoccludes the opening of the reservoir to prevent the liquid from flowinginto the analysis channel, is preferably a hydrophilic material such ashexadecanol or octodecanol. This advantageously promotes, rather thanobstructs the flow of liquid past any wax bits that harden within anyarea of the analysis channel after valve actuation. These materials alsopreferably have a melting temperature between 50 and 100 degreesCelsius, which allows for actuation with reasonable power-consumptionfor a battery-operated device, yet remains unactuated in generalhandling and storage environments and/or during a sonication protocol.In some embodiments, the amount of wax disposed upon the via is below 1microliter in its liquid state, and in some such embodiments, the amountis less than or equal to 0.5 microliters. In at least some embodiments,it is preferable to use as little wax as possible in order to reduce anyocclusion of the analysis channel and maximize full valve actuation whenheat is applied. In some embodiments, the valve also has afeedback-and-control system that allows for a consistent thermal profileto be achieved at the via for consistent valve actuation. Furthermore,this feedback-and-control system may incorporate sensing elements toenable the system to confirm that each valve has properly actuated.

In some non-limiting embodiments, the outlet at the bottom of thereservoir is sized and shaped, for example, as depicted in FIG. 20A orFIG. 20B. In FIG. 20A, the valve opening/outlet at the bottom of eachreservoir is depicted as a semicircle in fluidic communication with theanalysis channel. In some such embodiments, the semicircle has adiameter of approximately 1 mm, a size which may help reduce the amountof wax necessary to hold back the fluid of the reservoir from enteringinto the analysis channel. Alternatively, FIG. 20B depicts an outletformed of a semicircle with a boundary extension. In some suchembodiments, the boundary extension has a length between 0.1 mm and 1mm. Compared to FIG. 20A, the boundary extension of FIG. 20B may enhanceproper valve actuation and flow by providing a larger surface area forwax melted during the course of valve actuation to solidify onto beforeentering into the analysis channel. Such a configuration may reduce theamount of wax entering into the analysis channel. Similarly, duringvalve construction, the extensions from the semi-circle opening allowfor an increased area wherein the wax can harden without occluding theanalysis channel.

Returning to FIG. 18A, the electrical leads 754 are also electricallycoupled to the sensors 758; such an electrical connection allows signalsdetected by the sensors 758 to be delivered to the reader device forprocessing. In various embodiments, the sensors 758 and the area of theanalysis channel above them form the “analysis zone”, mentionedelsewhere herein. The sensors 758 are strategically located such that,when the circuit board 750 is included within the assembled cartridge700 with a surface of the circuit board 750 forming one wall of theanalysis channel 732, the sensors 758 are disposed within the analysischannel 732. As shown in FIG. 18A, a plurality of sensors 758 may beprovided, each spaced relative to the others, and all aligned with theanalysis channel 732. The sensors 758 are electrochemical sensors, eachforming an electrochemical cell within the analysis channel. In thisembodiment, each sensor 758 is formed of a working electrode 758 a, areference electrode 758 b, and a counter electrode 758 c. In otherembodiments, one or more of the sensors may be formed of only a workingelectrode. For example, in some embodiments, only one referenceelectrode and one counter electrode are provided within the analysischannel along with a plurality of working electrodes. In someembodiments, an oxidation reaction may occur at an electrochemicalsensor 758 if an oxidizing enzyme bound indirectly to a magneticparticle is present at the sensor 758 and an appropriate chemicalsubstrate is introduced into the analysis channel 732. In suchembodiments, the working electrode 758 a releases electrons to replenishelectrons stripped from the substrate by the oxidizing enzyme in aquantity proportional to the amount of oxidizing enzyme present. Therelease of electrons from the working electrode is a current which maybe detectable as a signal within a circuit connected to the sensor 758.The sensors 758 can thereby indirectly detect the presence, absence,and/or quantity of oxidizing enzymes localized in the analysis zone ofsuch embodiments. A computer, for example, within the reader devicedescribed below, can then correlate the presence, absence, and/orquantity of a target analyte to the presence, absence, and/or quantityof oxidizing enzymes. The functions of such a computer are described inmore detail below. In various embodiments, one or more magnetic fieldsare used to facilitate localization of the enzymes or other signalingagents within the analysis zone. Advantageously, in such embodiments, noaffinity molecules need to be pre-bound to the sensors to achievelocalization, which would otherwise significantly slow the analytequantification process due to the limits of diffusion-basedhybridization kinetics. Details of the magnetic fields are also providedbelow.

In some embodiments, the electrochemical sensors 758 where detectiontakes place are made through an electroless nickel immersion in gold(ENIG) process and thus have gold on the surface. In other embodiments,gold or gold-plated sensors are used that have not been made through anENIG process. In some embodiments, at least the working electrode 758 aof each sensor 758 has a surface chemistry formed of thiolated ethyleneglycol and/or a dithiol such as hexaethylene glycol dithiol for addedstability. The hydrophilic nature of the head groups of such surfacechemistry facilitates flow and protein resistance. Additionally oralternatively, in some embodiments, the surface of one or more of theelectrodes is backfilled with mercaptoundecanoic acid, mercaptohexanol,or other suitable backfiller. In some embodiments, the surface of one ormore of the electrodes within the sensor 758 is formed throughsequential addition and incubation of the ethylene glycol dithiol andthe backfiller at unelevated temperatures. In one embodiment, thesurface of the electrochemical sensors 758 includes a self-assembledmonolayer comprised of at least a mixture of an oligo-ethylene glycoldithiol (particularly hexa-ethylene glycol diothiol, tetra-ethyleneglycol dithiol, or other oligo-ethylene glycol dithiol) and a carboxylicacid terminated thiol. In one embodiment, the carboxylic acid terminatedthiol is mercaptoundecanoic acid with 11 carbons (i.e.,11-mercaptoundecanoic acid).

In at least some embodiments, each electrode within the electrochemicalsensors includes a filled hole via printed within the circuit board.While each electrode is printed on a top layer of the circuit boardcomponent at a location that places each electrode within the analysischannel, each via extends through the circuit board component and canthereby electrically connect the electrode on a top layer of the circuitboard component to traces located on another layer (e.g., a bottomlayer) of the circuit board component. A trace is a conductive track,such as a conductive metal track (e.g., a copper track), that provideselectrical communication between elements of a circuit board. The tracemay be provided to electrically connect the electrode to an electricallead of the circuit board component. The electrical lead is in directconnection with circuitry within a reader device (described below) whenthe cartridge is coupled to the reader device; accordingly, when thecartridge and reader device are coupled, the electrode is alsoindirectly connected electrically to the reader device. Thisconfiguration for circuit board elements may be useful in creating amore uniform surface on the top layer of the printed circuit board.Advantageously, a more uniform surface on the top layer improves theease with which the circuit board component (e.g., circuit boardcomponent 750) binds to the internal component (e.g., internal component730). If traces are instead provided on a top surface of the circuitboard component, the traces may interfere with the planarity of the topsurface and cause difficulties in bonding the internal component to thecircuit board component.

In various embodiments, one or more ambient electrochemical noisesensors, or reference sensors 759, are provided and spaced within theanalysis channel away from the site of magnetic particle localization.The reference sensor 759 with its associated circuitry quantifiesbackground noise in the system. Such noise may be due to, for example,the presence of non-specifically bound enzyme. In various embodiments,during processing of the detection results, a computer applies analgorithm to remove the reference sensor signal from the detectionsensor signal to account for and/or eliminate system noise and tothereby allow for proper quantification or detection of target analyte.

In some embodiments, the detection is carried out using a standardelectrochemical circuit that utilizes a bias potential generated at thereference electrode for the oxidation/reduction reaction to proceed. Thepotential is held at the reduction potential of the chemical substrate(low enough that there is little nonspecific reduction of reduciblespecies in the solution) so that the flow of electrons to the oxidizedmolecules can be quantified using an operational amplifier basedcurrent-to-voltage (op amp) circuit topology connected to the workingelectrode. For example, a common substrate molecule,tetramethylbenzidine (TMB), is used for HRP. When present, HRP oxidizesTMB molecules, and these molecules are in turn reduced by the workingelectrode. Since this event occurs in proportion to the amount of HRPpresent, a change in the current-to-voltage op amp measurement results.Using an analog-to-digital converter, the actual signal can be deliveredto a processor for processing. As described in more detail below, invarious embodiments, said processor and signal processing components areprovided within the reader device.

The Reader Device

The reader device, or reader, of various embodiments is, comprises, oris comprised of, a specialized computer. The computer includes aprocessor with memory having instructions stored thereon for executingone or more methods for detecting the presence, absence, and/or quantityof target analytes in a sample. In various embodiments, the reader'scomputer controls the operations of the detection system, controllingwhen and how various functions of the system occur, such as, forexample: mixing of the fluids in the first reservoir of the cartridge,opening of valves, and/or localization of magnetic particles over thesensors. To control such operations, the computerized reader isconfigured to receive information from, and send information to,physical components present within the reader or cartridge.

A functional block diagram of one embodiment of a reader is depicted inFIG. 21. Although described separately, it is to be appreciated thatfunctional blocks described with respect to the reader 2100 need not beseparate structural elements. For example, the processor 2110 and memory2120 may be embodied in a single chip. Similarly, the processor 2110 andcommunication interface 2150 may be embodied in a single chip. Invarious embodiments, the reader 2100 includes a power supply 2160 suchas a battery.

The processor 2110 can be a general purpose processor, a digital signalprocessor (DSP), an application specific integrated circuit (ASIC), afield programmable gate array (FPGA) or other programmable logic device,discrete gate or transistor logic, discrete hardware components, or anysuitable combination thereof designed to perform the functions describedherein. A processor may also be implemented as a combination ofcomputing devices, e.g., a combination of a DSP and a microprocessor, aplurality of microprocessors, one or more microprocessors in conjunctionwith a DSP core, or any other such configuration.

The processor 2110 is coupled, via one or more buses, to readinformation from, or write information to, the memory 2120. Theprocessor may additionally, or in the alternative, contain memory, suchas processor registers. The memory 2120 can include processor cache,including a multi-level hierarchical cache in which different levelshave different capacities and access speeds. The memory 2120 can alsoinclude random access memory (RAM), other volatile storage devices, ornon-volatile storage devices. The storage devices can include, forexample, hard drives, optical discs, flash memory, and Zip drives.

The processor 2110, in conjunction with software stored in the memory2120 executes an operating system, such as, for example, Windows, MacOS, Unix or Solaris 5.10. The processor 2110 also executes softwareapplications stored in the memory 2120. In one non-limiting embodiment,the software comprises, for example, Unix Korn shell scripts. In otherembodiments, the software can be programs in any suitable programminglanguage known to those skilled in the art, including, for example, C++,PHP, or Java.

The processor 2110 is also coupled to a cartridge interface 2130, whichmay include an EDGE card or other electrical connector, to sendelectrical signals to, and receive electrical signals from, the circuitboard component of the cartridge.

In some embodiments, the processor 2110 may be coupled to a userinterface 2140. For example, in some embodiments, the reader 2100 mayinclude a touchscreen, LED matrix, other LED indicators, or otherinput/output devices for receiving inputs from, and providing outputsto, a user. In other embodiments, the user interface 2140 is not presenton the reader 2100, but is instead provided on a remote computing devicecommunicatively connected to the reader 2100 via the communicationinterface 2150. Yet still in other embodiments, the user interface canbe a combination of elements on the reader and a remote computingdevice.

The communication interface 2150 of various embodiments is also coupledto the processor 2110. In some embodiments, the communication interface2150 includes a receiver and a transmitter, or a transceiver, forwirelessly receiving data from, and transmitting data to a remotecomputing device. In some such embodiments, the remote computing deviceis a mobile computing device that provides the system with a userinterface; additionally or alternatively, in some embodiments, theremote computing device is a server. In embodiments configured forwireless communication with other devices, the communication interface2150 prepares data generated by the processor 2110 for transmission overa communication network according to one or more network standardsand/or demodulates data received over a communication network accordingto one or more network standards. The communication interface 2150 ofsome embodiments may additionally or alternatively include electricalconnections for wired communication of signals between the reader 2100and a remote computing device.

In addition to the computing components, the reader of variousembodiments, includes several additional physical components needed toimplement target analyte detection. For example, the reader 2200 of FIG.22 includes a slot, opening, bed, port, or other docking feature,referred to herein as a dock 2210, for receiving a cartridge. Thecartridge, when received by the reader 2200, may be disposed on or in,or otherwise coupled to, the reader 2200.

Several of the reader components are strategically positioned inparticular locations relative to the dock 2210 in order to achievedesired interactions with the cartridge. For example, the reader 2200 ofthe depicted embodiment includes an electrical connector 2220 and one ormore magnetic field generators 2240, and the location of such componentsis selected to align with particular features of a docked cartridge.Additionally, some embodiments, including the embodiment of FIG. 22,include a sonication element 2230. Each of these components is describedin more detail below.

The electrical connector 2220 of various embodiments is an EDGE card orother connector having pins for electrical connectivity. The connector2220 is located on, under, within, or adjacent to the dock 2210 and ispositioned such that the pins of the connector 2220 make contact with,and establish electrical connectivity with, the electrical leads of adocked cartridge device. The electrical connector 2220 therebyestablishes electrical continuity between the sensors on the circuitboard component of the cartridge and electrochemical circuitry withinthe reader. In some embodiments, the electrical connector 2220 of thereader may also establish electrical continuity with a heating element,if present on the circuit board component of the cartridge. In someembodiments, the reader 2200 includes a portion of an electrochemicalcircuit, which is completed with the addition of the cartridge based onelectrical continuity between the electrical connector 2220 and theelectrical leads of the cartridge. In such embodiments, the addition ofthe cartridge completes or closes the circuit. In such embodiments,coupling the cartridge to the reader 2200 activates the reader, causingit to “wake up.” Once awoken, the electrical connector 2220 may identifysignals being received from a portion of the cartridge to identify whattype of cartridge is coupled to its dock. In some embodiments, theelectrical connector 2220 may identify a label, such as, for example, aresistive label on the cartridge, which is unique to a particularcartridge type in order to identify the docked cartridge type. In otherembodiments, a digital barcode coded within the electrical leads of thecircuit board component of the cartridge is read by electrical pins orpads within the reader to identify the cartridge type. In some suchembodiments, the circuit board component of the cartridge includes aplurality of electrical leads, some of which are connected to a groundlead and some of which are not. Through a combinatorial usage of theelectrical pins and connections between them and a ground pin, and/orwith a pull-up/pull-down resistor located on the reader, the condition(e.g., grounded or not grounded) of each pin is sensed as a higher orlower voltage than a set voltage, which is read as a logic situation atthe processor of the reader to determine whether a particular pin isgrounded. In this manner, a combination of grounded and non-groundedpins can be detected and recognized by the reader 2200 to uniquelyidentify classes of cartridges.

In some embodiments, once awoken, the reader 2200 also determines whattest protocol to run for the identified cartridge and/or searches for,and connects to, nearby mobile computing devices.

Continuing with FIG. 22, the reader 2200 optionally includes asonication component, or sonicator 2230. The sonicator 2230 of variousembodiments is located in, under, or over the dock 2210 and ispositioned directly or substantially over or under the first reservoirof a docked cartridge. In some embodiments, the docked cartridgeincludes features to facilitate a close relationship between thesonicator 2230 and the first reservoir. For example, as seen in FIG. 7B,the circuit board component 750 and base component 740 are each shapedto provide a cutout or window 741, 751 in their structures, the cutouts741, 751 aligned with the reservoirs. Thus, in various embodiments, thesonicator 2230 and the first reservoir of the cartridge can be alignedwith no structures provided between them. In some embodiments, when theuser slides the cartridge into the dock, the cutouts 741, 751 allow forthe sonicator 2230 to be positioned directly underneath the reservoir.Such a configuration enables the sonicator 2230 to transmit controlledamounts of energy into the first reservoir. In other embodiments, thesonicator (or other component performing the sonication steps disclosedherein) is disposed on or forms a bottom wall of the first reservoir724, such as, for example, as shown in FIGS. 11A-11C. In otherembodiments, no sonicator is provided. In various embodiments having asonicator 2230, the sonication energy is controlled to achieve mixingand hybridization of components within the first reservoir whilelimiting damage caused to fragile DNA probes or other molecules.

In some embodiments, the sonicator 2230 includes a pressure-sensitivepiezoelectric disk 2232. Optionally, in some embodiments, the sonicator2230 further includes a high water content blister disposed between thefirst reservoir and the piezoelectric disk. In some embodiments, thehigh water content blister is affixed under the first reservoir in thecartridge production process; in other embodiments, it is provided overthe sonicator 2230 within the reader. The high water content blister mayfacilitate delivery of sonic energy from the sonicator 2230 to the firstreservoir with minimal attenuation. In some embodiments, the blister isreplaced with another appropriately conducting sonication medium. Insome embodiments, the component serving as a sonication medium ispreferably dry on the outside, with no liquid residue present. In someembodiments, when the cartridge slides into the reader 2200, thesonically conducting medium coupled to the sonicator forms a soft sealwith a sonically conducting medium affixed to the bottom of the firstreservoir. This “soft seal” may be enhanced by using a conformalsonically conducting medium on the bottom of the reservoir.

In addition to generating sonic energy to mix and hybridize the contentsof the first reservoir, in various embodiments having a sonicator 2230,the sonicator 2230 can be used to detect the introduction of the samplecollection device into the first reservoir. Advantageously, suchdetection enables the reader 2200 to initiate an automated start of atesting protocol immediately or substantially immediately followingintroduction of a sample into the first reservoir. The automated startimproves ease of use for lay users; it also ensures a consistent starttime relative to sample introduction, thus providing consistent results.

As mentioned above, in some such embodiments, the sonicator 2230 is apressure-sensitive piezoelectric element. In such embodiments, a wall ofthe cartridge is designed to flex slightly upon insertion of the samplecollection device into first reservoir; such flexing results in a changein pressure, which is detectable by the sonicator 2230.

In other embodiments, detection of the sample collection device in thefirst reservoir occurs through resonance or signal monitoring.Specifically, as shown in FIG. 23A, upon activation of the reader, forexample, as a result of coupling a cartridge to the reader, thesonicator 2300, and/or the piezoelectric element forming all or aportion of the sonicator 2300, generates a sound wave 2310 directedtowards the first reservoir. In some embodiments, the sonicator 2300 ora portion thereof then deforms as a result of a reflected sound wave 315and/or the resonance frequency of the sonicator is recorded, therebyallowing a processor and/or circuit within the reader to determine abaseline, unloaded condition. Thereafter, the sonicator 2300 enters ascanning condition shown in FIG. 23B, periodically pinging a sound wave2310 into the first reservoir so the processor or circuit within thereader can monitor the return signal 2315 and/or resonance frequencyshift of the sonicator to determine if any variation has occurred. Insome embodiments, if no variation has been detected and/or the baselineunloaded condition is being established, the reader emits one or morelights or sounds prompting a user to enter a sample collection deviceinto the cartridge. At FIG. 23C, the addition of the sample collectiondevice 2350 causes a shift in resonance of the sonicator and/or a changeto the sound wave return signal above a threshold which the processor orcircuit within the reader is programmed to identify as sample collectiondevice insertion. In various embodiments, the processor and/or circuitthen returns instructions to the sonicator 2300 to initiate a sonicationstep of the testing protocol. In some embodiments, a light pattern onthe reader changes or a sound is emitted to signify that a testingprotocol has been initiated. In some embodiments, the user-promptinglight or audible user-prompting sound pattern emitted during thescanning phase experiences a change in intensity or frequency to signalincreased urgency to the user to input a collection device. In variousembodiments, the sound waves associated with prompting the user aredistinct from the sound waves emitted by the sonicator to establish theloaded vs. unloaded condition of the reservoir.

In some embodiments, a high intensity sonication procedure is performedto actively elute the sample particles, including, if present, thetarget analyte, into the solution of the first reservoir. The sonicationprocedure is also performed to achieve proper suspension of the samplepreparation reagents, particularly the magnetic particles, in order tomake the magnetic particles available in solution for binding with thetarget. Any sonicator may be used which is capable of achieving the goalof generating a gentle sonication, even at the high intensity phase,while avoiding cavitation and large shearing forces. One embodiment ofan appropriate sonicator is a piezoelectric component, such as, forexample, a 1.6 megahertz bending transducer piezoelectric disk at anoutput of less than 15 Watts.

Following the high intensity sonication, the sonic signal of thesonicator is pulsed in order to prevent the magnetic particles fromsettling and to continue to add energy into the system. The addition ofenergy enhances the hybridization between the affinity molecules on themagnetic particle, the target, and the detector agent or competitivebinding agent.

The sonication profile selected by the reader varies according to thesample being tested. As used herein, “sonication profile” refers tocharacteristics of the delivered sonication, such as the length of timeof sonication, the frequency of sonication, the intensity, etc. Invarious embodiments, the reader has fine-grained control over suchvariables. In some embodiments, for power consumption purposes, thesonicator has an “on period” in which it pulses. For example, in oneembodiment, during the sonication phase, the sonicator is activated forthree seconds within every 10 second window, and within those threeactivated seconds, the sonicator pulses at regular intervals; forexample, the sonicator may generate a sound wave every 0.027 seconds.Such methods create an environment conducive to hybridization, targetcapture, and formation of various molecule complexes while avoidingover-consumption of power and over-heating of the sample.

Continuing with FIG. 22, the reader 2200 of various embodiments alsoincludes one or more magnetic field generators 2240. In some embodimentsthe magnetic field generator 2240 may be an inductor or otherelectromagnetic component affixed within the reader 2200. As shown inFIG. 22, in some embodiments, the magnetic field generator 2240 is apermanent magnet. The magnetic field generator(s) 2240 are positionedsuch that, when a cartridge is coupled to the dock 2210, the one or moredetection sensors are each disposed directly within a magnetic fieldcreated by the magnetic field generator(s) 2240. In various embodiments,the magnetic field(s) are the cause of localization; the magneticfield(s) are what induce magnetic particles and accompanying hybridizedmolecules to localize within the analysis zone.

In various embodiments, the base component of the cartridge has a cutoutthat allows for at least one permanent magnet or inductor to bepositioned directly underneath the detection sensor of the circuit boardcomponent. The cutout allows the cartridge to slide into place on thedock 2210 without hitting the magnet or inductor. The cutout also allowsfor the magnetic field generator 2240 to be positioned as close to thedetection sensor as possible. The closer the magnet field generator 2240is to the sensor, the more force the magnet field is able to exert,meaning that smaller magnets or inductors are capable of exertingequivalent magnetic field strengths as larger, more costly magnets orinductors. The use of small magnets or inductors is particularlyadvantageous in embodiments having multiple magnetic fields and multipleanalysis zones (for example, in embodiments configured to detect aplurality of different target analytes), because the smaller the magnetor inductor, the less the magnetic fields overlap. Smaller magneticfields can limit the amount of cross talk between the magnets orinductors under the different detection sensors.

Additionally, as mentioned above in the discussion of the cartridge, insome embodiments of an analyte detection system, a heating element isprovided to activate heat-actuated valves within the reservoir outlets.In such embodiments, the heating element delivers heat to vias on thecircuit board component of the cartridge, and the vias act as conductorsof heat to melt wax at precise points of time and/or within precisespatial area. In some embodiments, a plurality of heating elements arelocated within the reader 2200 and positioned to align with the vias ofa docked cartridge. In some such embodiments, spring-loaded contacts areprovided within the reader 2200 to form an effective contact between theheating elements of the reader and the via. In some such embodiments,the heating element is a resistive heating element.

In various embodiments, regardless of the location of the heatingelement (in the reader or the cartridge), the timing of heat deliveryand valve opening is precisely timed and controlled by the readerdevice. For example, in some embodiments, the reader computer controlswhen heat-generating current flows through the heating element. Thevalves are actuated by heat caused by such current in the followingsequence: (1) sample preparation reservoir, (2) wash reservoir, ifpresent, then (3) chemical substrate reservoir. Actuation of each valveis timed such that: the respective valve fully actuates, the associatedreservoir has time to empty its contents into the analysis channel, andat least some of the contents of the reservoir have time to travel tothe absorbent pad positioned downstream of the sensors before thecontents of the next reservoir is released. In some embodiments, thetime between valve actuations is selected to be great enough for theabsorbent pad to entirely or substantially absorb liquid present withinthe analysis channel. Advantageously, in such embodiments, very littlemixing occurs between the contents of successive reservoirs.

In some embodiments, the precise timing of sequential valve actuationand/or determination of successful valve actuation can be determined atthe processor through the usage of feedback control systems utilizing analgorithm on the processor and information derived from sensingelements, such as thermistors and electrochemical sensors. For example,an electrochemical sensor in the analysis channel can be queried todetermine whether the analysis channel has liquid in it since the signalgenerated at the sensor will be different depending upon the presence orabsence of liquid above the sensor. This signal, in combination with aprocessor set to logically interpret the signals, can thereby determinewhether a valve has actuated properly and/or when a reservoir has fullyemptied its liquid contents and when the liquid contents have beenabsorbed into a waste pad such that the channel is free of liquid andready for a subsequent valve actuation. In some embodiments, theprocessor and/or circuitry of the reader sends signals instructing aheating element to actuate a subsequent valve only after the processorand/or circuitry has received confirmation, through the feedback system,that the analysis channel is wholly or partially cleared and ready forthe next step.

Additionally, as shown schematically in FIG. 24, in some embodiments, adesired thermal profile of the heating element for valve actuation canbe consistently achieved through the usage of an additional feedback andcontrol system 2400 which includes: a temperature sensing element 2410,such as a thermistor, in thermal communication with a heating element2420 positioned to actuate a heat-actuated valve 2430, and a processor2440 set to logically interpret signals from said heating element 2420.

One embodiment of a thermal profile-controlling feedback and controlsystem is provided in FIG. 25. Alternate FIG. 25 depicts an embodimentof a temperature sensing element 2510 in thermal communication with thevia 2520 on a circuit board component 2530 of a valve actuating element.In particular, the temperature sensing element depicted is a thermistor,which has a resistance that varies with its temperature. When inelectronic communication with other circuitry and/or a processorconfigured to interpret the electronic signals from the thermistor, theinformation gathered from the thermistor can be utilized to maintainconsistent thermal actuation of the valve through command and control ofa heating element in electronic communication with aforementionedprocessor. This sensing element can additionally improve safety of thesample analysis device by helping to prevent runaway escalation oftemperature at the heating element in thermal communication with saidthermistor by contributing sensing information that will enable aprocessor to shutoff the heating element if the temperature runs toohot. The depicted embodiment of FIG. 25 shows the thermistor 2510 inthermal communication with the heating element through the usage of aheat conducting element on a circuit board of the reader device, saidheat conducting element being a metallic trace 2540, for example, acopper trace. Additionally, the thermistor 25510 is in thermalcommunication with the via 2520 of the valve unit through the use of aconnector 2550 (in one embodiment, a spring loaded connector pin), whichtypically has high thermal conductivity. The heating element is notdepicted in FIG. 25, but it can be appreciated that the heating elementcan be thermally coupled to the via in multiple ways, including throughthe usage of a trace as the thermistor is coupled to the via through aconducting trace and then through the spring loaded pin in contact withthe via.

FIGS. 26A-26C depict the reader device 2200 of FIG. 22 shown throughvarious stages of coupling to a cartridge 700. As shown, the reader 2200includes a dock 2210, an electrical connector 2220, a sonicator 2230,and a magnetic field generator 2240 in the form of a permanent magnet.The cartridge 700 is configured to slide into the dock 2210 and coupleto the reader 2200. When coupled, the electrical leads 754 of thecartridge 700 are in direct contact with the electrical connector 2220,the first reservoir 724 is disposed over the sonicator 2230, and aportion of the microfluidic analysis channel 732 is disposed over themagnetic field generator 2240 within the magnetic field.

FIGS. 27A and 27B provide an additional embodiment of a reader device2700 coupled to a cartridge 2702. The reader device of FIGS. 27A and 27Bincludes a plurality of magnets 2740 disposed in series below the dockof the reader 2700, positioned such that when a cartridge 2702 iscoupled to the dock, the magnets 2742, 2744, 2746, 2748 are locatedbelow a plurality of detection sensors 2762, 2764, 2766, 2768,respectively. In embodiments such as the embodiment of FIGS. 27A and27B, which are designed to detect the presence, absence, and/or quantityof a plurality of different target analytes in a sample, modificationsare made to both the design of the cartridge 2702 and the reader 2700relative to other embodiments described herein. For example, asdescribed above, the detection of multiple different target analytesrequires the inclusion of multiple populations of magnetic particles andmultiple populations of detector agents and/or competitive bindingagents within the first reservoir 2724. Each population of magneticparticles, detector agents, and competitive binding agents present inthe reservoir 2724 is designed to have affinity to a different targetanalyte and include a different capture antibody, capture DNA probe orother affinity molecule. Additionally, each population of magneticparticles present in the reservoir 2724 has a unique identifyingphysical characteristic, such as a different size, magnetic response,density, or any combination thereof.

In one embodiment in which multiple populations of magnetic particlesare present to detect the presence of a plurality of different targetanalytes, dead-end filtration is used to separate the populations fordetection. In such an embodiment, as the magnetic particles flow out ofthe first reservoir 2724 and into the analysis channel 2704, a sequenceof filters provided within the analysis channel 2704 are encountered.Moving in a downstream direction, the filters are ordered by pore sizewith the first filter having the largest pores and the last filterhaving the smallest pores. Each filter is placed in close proximity to adetection mechanism designated to detect a particular detector agent ora product of a particular detector agent. For example, in someembodiments, the detection mechanism is an electrochemical sensordesignated to detect oxidation that occurs among a particular populationof hybridized magnetic particles. Magnetic particles smaller than thefirst filter pore size will pass through the filter with the flow ofliquid down the channel. Magnetic particles larger than the pore size ofthe first filter will remain behind, in close proximity to the firstsensor 2762. Through the use of successive filters of decreasing poresize, the magnetic particle populations are separated and localized overthe different detection sensors 2760. In this manner, reactions such asoxidation reactions among different populations of hybridized magneticparticles and target analytes can then be monitored in the mannerdescribed elsewhere herein to identify the presence, absence, and/orquantity of each of a plurality of target analytes.

This process can be enhanced through the use of magnetism. Magneticparticles of the same material composition vary in their magneticresponse with the square of the diameter of the magnetic particle.Therefore, a magnetic field will interact differently on magneticparticles of different size, thus allowing a sorting mechanism to takeplace. This differential magnetic response may be exploited in someembodiments to enhance separation speed and specificity. As the magneticparticles leave the first reservoir, a magnetic field may be applied tothe analysis channel in order to at least partially order the magneticparticles by size. Since larger magnetic particles will feel themagnetic force more strongly than smaller magnetic particles, the largerones will move more slowly downstream relative to the smaller magneticparticles. This results in a preference for smaller magnetic particlesto progress down the analysis channel 2704 earlier than larger magneticparticles, which decreases the likelihood of magnetic particle-basedclogging of pores. Magnetic particle-based clogging of a pore maydecrease multiplexing specificity and may prevent proper testingaltogether by restricting the flow of liquid needed to wash away excessenzyme and to provide chemical substrate to the captured detectoragents.

In some embodiments, cross flow filtration technology is used to preventmembrane fouling common with dead-end filtration. In such embodiments,the magnets or inductors are positioned to exert a perpendicular orother non-parallel magnetic force relative to the direction of flow.Such a placement of the aligning magnets or inductors causes magneticparticles to be pulled to the side of the analysis channel where thefilters are located, if they are of sufficient size to be acted upon bythe provided magnet field generator. In such embodiments, the magnetfield sizes are selected such that a magnetic particle will be pulled tothe side of the analysis channel 2704 to encounter a filter justupstream of the first filter having a pore size smaller than the size ofthe magnetic particle.

Alternatively, the populations of magnetic particles and target analytescan be separated through the use of magnetism alone. Because themagnetic force response of a magnetic particle scales with the square ofthe diameter of the particle, separation and localization of magneticparticle populations can be achieved in a single channel without the useof membranes by providing a plurality of magnets or inductors located onthe reader device or cartridge creating different magnetic fieldstrengths at different locations of the analysis channel 2704.Specifically, moving in a downstream direction, magnetic fieldgenerators of increasing magnetic field strength are encountered. Thelargest magnetic particles are localized at the first sensor becausethey are unable to escape the first magnetic field, which is just strongenough to capture the largest magnetic particles, but is not strongenough to capture any other size of magnetic particles. Magneticparticle populations will travel downstream with the flow of liquiduntil they are caught by the magnetic field tailored for theirparticular magnetic particle size located over a detection sensor 2760provided for detecting oxidation reactions among their population. Thesecond weakest magnetic field will capture the population of magneticparticles with the second largest diameter; the third weakest magneticfield will capture the population of magnetic particles with the thirdlargest diameter, and so on. The smallest magnetic particles arecaptured by the strongest magnetic field. This allows each population ofmagnetic particles to localize over a different detection sensor anddetection proceeds as described above. The magnetic fields can be variedthrough at least a couple methods. In some embodiments, each magnet orinductor is a different size; the larger the magnet or inductor, thelarger its magnetic field. In other embodiments, such as the embodimentshown in FIGS. 27A and 27B, magnets 2740 are placed at varying depthsrelative to the plane of the analysis channel 2704. The upstream-mostmagnet 2748, is placed furthest to the analysis channel 2702, and thusexerts the strongest magnetic field on the channel 2704. Thedownstream-most magnet 2742, is placed closest to the analysis channel2702, and thus exerts the strongest magnetic field on the channel 2704.

Importantly, in various embodiments described herein, the magneticattraction between the magnetic particles and the one or more magnetfields is sufficiently strong to cause the magnetic particles to remainlocalized over the one or more magnetic field generators as a washsolution and/or a liquid carrying chemical substrates flows over themagnetic particles.

The Detection System

One embodiment of a detection system 2800, which includes the samplecollection device 400 of FIGS. 4A and 4B, the cartridge device 700 ofFIGS. 7A and 7B, and the reader device 2200 of FIG. 22, is provided inFIGS. 28A and 28B. The devices forming the system are shown separately,prior to use, in FIG. 28A and in a coupled configuration, in use, inFIG. 28B. The sample collection device 400 of various embodiments,including the embodiment of FIG. 28A, is disposable and configured forone-time use. It may come within removable sterile packaging. Onceinserted into the input tunnel 712 of the cartridge 700, the samplecollection device 400 is locked into a permanent fixed engagement andcannot be used again. Similarly, the depicted cartridge 700 isdisposable and configured for one-time use. Once the sample collectiondevice 400 locks into place within the input tunnel 712 of the cartridge700, the cartridge 700 cannot be used again. The cartridge 700, can,however, be removed from the reader 2200. In various embodiments, thecartridge 700 and the reader 2200 are configured to be separablycoupled, and the cartridge 700 can be inserted and removed from the dockof the reader 2200 at least before and after implementation of adetection protocol. In some embodiments, the reader 2200 may include alocking mechanism for temporarily locking the cartridge 700 into place,and limiting removal, during the duration of a detection test cycle. Thereader 2200 of various embodiments is reusable.

Additionally, in certain embodiments, the reader 2200, and the entiredetection system 2800, are configured for non-clinical,consumer-directed use. Accordingly, the system 2800 of some embodimentsis easy to use and generates results quickly. In some embodiments,results of a target analyte detection protocol are generated in 30minutes or less from the time a sample from a sample collection device400 is inserted into the system's cartridge 700. In some embodiments,the results are generated in less than 20 minutes, in some embodiments,less than 10 minutes, and in some embodiments, results are generated inless than 5 minutes. Additionally, the consumer-directed system of someembodiments is small for an unobtrusive presence within a home, school,office, or other place of employment. In some embodiments, the system isless than 30 cm in height, less than 30 cm in width, and less than 30 cmin length; in some embodiments, the height, width, and length are eachless than 20 cm; in some embodiments, one or more of the height, width,and length are less than 10 cm. In some embodiments, the cartridge 700,sample collection device 400, and reader 2200 together form a system2800 approximately the size of a smartphone or other mobile computingdevice. In some embodiments, the system is sized and configured to beportable. In such embodiments, in addition to a compact, hand-helddesign, all liquids within the sample are properly sealed and separatedsuch that no leaking or premature oxidation reactions will occur due tojostling of the system components while on the go.

To promote use by lay people in non-clinical settings, the system 2800of some embodiments is designed to be “dummy proof” by including aself-activating and self-run detection protocol. For example, FIG. 28Bdepicts an example in which the cartridge 700 has been placed into thedock 2210 of the reader 2200 and the sample collection device 400 hasbeen inserted into the input tunnel 712 of the cartridge 700. In thedepicted embodiment, loading the cartridge 700 into the reader 2200established an electrical connection between the pins of the cartridge700 and the reader 2200, thereby completing a circuit within the reader2200, which automatically activated the reader. Upon being activated,the reader 2200 of some embodiments activates its sonicator, if present,utilizing the sonicator to detect entry of a sample collection device400 into the first reservoir. Upon detection, the reader 2200 of variousembodiments is configured to initiate a detection protocol automaticallywithout any further human intervention. The automated start ensures thatmixing of reagents and sample within the first reservoir occursconsistently at a fixed time following insertion of the samplecollection device, leading to consistent test results. In otherembodiments, where no sonicator is present, the testing protocol mayinitiate when a user presses a “go”, “run”, “start”, or other similarbutton or icon on the reader 2200 or a remote computing device 2820.

As described in more detail below, and as shown in FIGS. 28A and 28B, insome embodiments, the system 2800 includes a remote computing device2820. The remote computing device 2820 may be a mobile computing device,such as, for example, a smartphone, tablet, or wearable device, or alaptop or other computer. As shown in FIG. 28A, in some embodiments, thereader 2200 communicates with the remote computing device 2820wirelessly. In other embodiments, a removable wired connection, such asa cable connection, is provided between the reader 2200 and the remotecomputing device 2820. In still other embodiments, such as theembodiment of FIGS. 29A and 29B, an analyte reader 2910 having acartridge docking station 2915, within the system 2900, removablycouples to the remote computing device 2920 directly, for example, byconnecting via a plug 2912 into a headphone jack or electrical chargingport.

In various embodiments, the remote computing device may be includedwithin the system: to provide for more computing power and/or morememory; to provide a wireless transceiver for pulling data from, andtransmitting data to, a remote server; and/or to provide a displayscreen and user interface. A remote computing device is not neededwithin every embodiment. For example, as shown in FIG. 30, in someembodiments, the reader 3000 includes a processor and memory (notshown), a dock 3015 for a cartridge, as well as a touchscreen or otheruser interface 3010. In such embodiments, the reader is configured toidentify the proper test protocol, run the test protocol, analyze theraw results received from the sensors in the system, and display digitalresults to a user. The reader of such embodiments may further include awireless receiver and transmitter for accessing and transmitting datafrom remote servers.

One embodiment of an analyte detection system is shown schematically inFIG. 31. FIG. 31 provides a schematic illustration of the interactionsbetween computerized components within one embodiment of an analytedetection system 3100. One skilled in the art will appreciate that theembodiment is illustrative in nature only and various components may beadded, deleted, or substituted and various different hierarchies andmodes of communication between the devices may be employed. In thedepicted example, the detection system 3100 is formed of a plurality ofcomputerized devices, including a reader 3130, a device having a userinterface 3140, and a server 3150. While not computerized, the system3100 additionally includes a sample collection device 3110 and acartridge 3120 shown coupled to the reader 3130. It should be understoodthat in certain embodiments described with reference to FIG. 31, thereader 3130 may represent any reader embodiment described elsewhereherein, such as for example, reader 2200, reader 2910, or reader 3000.Similarly, the device having a user interface 3140 may represent anysuch device described herein, such as the mobile computing device 2820or 2920. The cartridge 2820 may represent any cartridge embodimentdescribed herein, such as cartridge 700, 800, or 900 and the samplecollection device 2810 may represent any sample collection devicedescribed herein, such as sample collection device 400 or 600. Thesystem 3100 includes a communication network 3160 through which some orall of the various devices communicate with one another. The network canbe a local area network (LAN) or a wide area network (WAN). In someembodiments, the network is a wireless communication network, such as,for example, a mobile WiMAX network, LTE network, Wi-Fi network, orother wireless network. In other embodiments, the communication betweenthe computer having a user interface 3140 and the server 3150 occursover the internet via a wired network, such as a DSL cable connection.

In some embodiments, the reader 3130 and the device having a userinterface 3140 are not separate devices, but rather, are both providedwithin the reader device 3130, for example, as shown in FIG. 30. In suchembodiments, communication between the reader processor and the userinterface occurs internally within the reader 3130 via the transmissionof electrical signals.

In other embodiments, the reader 3130 and the device having a userinterface 3140 are separate devices. In some embodiments, the devicewith the user interface 3140 is a smartphone or other mobile computingdevice. Communication between the reader 3130 and the mobile computingdevice 3140 may occur, wirelessly, for example, using Bluetooth®,near-field communications, or other radiofrequency technology.Alternatively, transmission of signals between the reader 3130 and themobile computing device 3140 may occur over a cord, cable, or otherwired or direct connection. In various embodiments, the mobile computingdevice or other device having a user interface 3140 includes a softwareapplication for a front-end, graphical user interface for presentingtest results to a user.

In various embodiments, the reader 3130 is configured to control thetests and processes needed to detect and/or quantify target analytewithin a sample. To do so, a significant amount of information may bestored within the memory of the reader 3130. Alternatively, some or allof the information may be stored within the server 3150 and accessibleby the reader 3130 via the communication network 3160. Such informationincludes, for example a database of cartridge keys, which identifieseach cartridge type by the signal generated by the cartridge's uniqueidentifying resistor label. The information also includes test protocolsassociated with each cartridge key. The test protocols may specify suchdetails as how long to mix sample preparation reagents throughsonication, the frequency of the sonication, when to heat the variousheat-sensitive valves, etc. The information may also include correlationtables for each cartridge type, which correlate detected sensor signalsto the absence, presence, and/or a specific quantity of a targetanalyte. Additionally, the information stored by the reader 3130 and/orthe server 3150 may include one or more past results. In someembodiments, the reader 3130 stores test results at least until thereader 3130 comes into communication with a remote computing device; atsuch time, the results may be transmitted to the remote computing device(mobile computing device 3140 or server 3150) for display and/orlong-term storage.

In some embodiments, the server 3150 also stores user profiles, whichmay include biographical information entered into the system by a userthrough the device having a user interface 3140. In some suchembodiments, a log of test results for each user is also stored by theserver 3150 and accessible for viewing by the user through transmissionof such data to the device with a user interface 3140.

In one embodiment, when a cartridge 3120 is loaded into the reader 3130,the reader 3130 detects signals from a label, such as a resistor labelor electronic barcode, on the cartridge 3120 to detect the cartridgetype. The reader 3130 compares the detected signals to a database ofknown label signals or cartridge keys to determine which cartridge typeis present. If the detected label signal is not found within thedatabase of cartridge keys, the reader 3130 may transmit a message to aserver 3150 requesting updates to the database of cartridge keys. Thereader 3130 may transmit the message directly to the server 3150 orindirectly by way of the mobile computing device 3140. The reader 3130may additionally receive, directly or indirectly, data for cartridge keydatabase updates. The data may include new cartridge types and thecartridge keys and test protocols corresponding to each new cartridgetype. In some embodiments, the reader 3130 then identifies andimplements the test protocol associated with the detected cartridgetype. Upon receiving signals from a detection sensor, the reader 3130 ofsome embodiments compares the signals to a correlation table to processthe signals and generate meaningful results. The results may betransmitted to the device with a user interface 3140 for display to auser. One skilled in the art will appreciate that the variousinformation stored by the computing devices of the detector system 3100may be stored by any one or more of the devices and may be accessible tothe other devices through the receipt and transmission of data signals.

The Computerized Methods of Detection

As mentioned above, the computerized reader largely controls theoperations of the detection system. The reader includes a processor andmemory, the memory having instructions stored thereon for implementingvarious methods needed to successfully detect the presence, absence,and/or quantity of target analyte within a collected sample. Forexample, an embodiment of one method performed by the computerizedreader in an automated manner is provided in FIG. 32.

At block 3202, the computerized reader detects the presence of acartridge loaded into or onto the reader. For example, in someembodiments, a cartridge is coupled to the reader such that electricalleads on the cartridge come into physical contact with electrical pinson the reader, completing a circuit that turns on the reader and signalsthe reader to the presence of a cartridge.

At block 3204, the reader detects identification information associatedwith the cartridge. For example, the cartridge of some embodimentsincludes a unique identification key on its circuit board component,which generates signals unique to the particular cartridge type of thecartridge, allowing the reader to distinguish between cartridge types.The identification key may be a resistive element, for example, asurface mount resistor or a resistive ink-based element having a uniquesize or shape, or it may be another unique electrical signal generator.

The reader's processor receives the unique identification key signalsfrom the reader's circuitry which detected the signals, and as shown atblock 3206, identifies a proper test protocol for the cartridge based onthe unique identification key. In some embodiments, the reader'sprocessor compares the unique identification key signals to a databaseof identification keys stored in memory. Within the database of someembodiments, each identification key is associated with a particularcartridge type and test protocol. If the identification key signalsreceived from the processor match a key in the database, thecorresponding test protocol will be opened and executed by theprocessor. If the identification key signals do not match a key in thedatabase, the processor may communicate with a remote computing devicesuch as a mobile computing device and/or a server to signal that anunidentifiable cartridge has been detected. In some embodiments, thereader downloads updates directly from a server or indirectly with themobile computing device acting as an intermediary. In some embodiments,when an unknown cartridge type is detected, a user is prompted via theuser interface of the mobile computing device, to download updates; inother embodiments, the updates are downloaded automatically. In variousembodiments, the updates include newly developed cartridgeidentification keys and test protocols. Once the new identification keysand test protocols are downloaded, they will be added to the reader'sdatabase of supported tests so that future tests with this cartridgetype will automatically be recognized and implemented without the needfor communicating with remote computing devices.

As shown at block 3208, in various embodiments, the computerized readerdetects insertion of a sample collection device into a first reservoirof the cartridge. Various processes can be implemented to accomplishthis detection, as provided in more detail in the discussion ofsonication above. In various embodiments, the reader's processorreceives signals from a sonicator element comprised partially or whollyof a piezoelectric element, in the reader. By monitoring the sonicatorelement to identify changes in the signals generated from a mechanicalevent within the reservoir, the processor can identify when a change inpressure and/or a change in resonance and/or a change in a reflectedsignal (pressure or sound wave) has occurred in the first reservoir ofthe cartridge through the ability of the piezoelectric component totransduce mechanical signals into electric signals which can beamplified and understood through a combination of circuitry andprocessor in electronic communication with said piezoelectric element;such changes are indicative of entry of a sample collection device intothe reservoir.

At block 3210, the reader's processor sends signals to the sonicator toinstruct it to initiate a sonication protocol to mix a plurality ofreagents, affinity molecules, and sample particles within a liquiddisposed within the first reservoir. In various embodiments, theresulting mixture includes magnetic particles bound to: target analytes,target analytes and detector agents, and/or competitive binding agents.As used herein, sandwich complexes refer to magnetic particles bounddirectly or indirectly to target analytes and detector agents;competitive binding complexes refer to magnetic particles bound tocompetitive binding agents. Each sandwich complex and competitivebinding complex include a detector agent bound within the complex. Inone embodiment described here, the detector agent is an oxidizingenzyme.

As shown at block 3212, in some embodiments, the reader generates acurrent, which heats or otherwise stimulates a first heating element,thereby causing heat to transfer to a first heat-actuated valve withinthe cartridge. In some embodiments, this causes the valve to melt orundergo another phase change, which allows liquid to flow out of thefirst reservoir into an analysis channel via capillary action. As theliquid flows, it transports the mixture with it, and the magneticparticles within the mixture, including magnetic particles withinsandwich complexes and/or competitive binding complexes, localize overone or more magnetic fields within the analysis channel, forming one ormore localized samples.

Optionally, at block 3214, the reader generates a current, which heatsor otherwise stimulates a second heating element such that a secondvalve within the cartridge undergoes a phase change and a wash solutionflows out of a second reservoir into the analysis channel. In variousembodiments, the wash solution removes, from the one or more localizedsamples, oxidizing enzymes (or other detector agents) that are notindirectly bound to magnetic particles.

At block 3216, the reader generates a current, which heats or otherwisestimulates a third heating element such that a third valve within thecartridge undergoes a phase change and a solution of substrates flowsout of a third reservoir into the analysis channel. In variousembodiments, when the detector agent is an oxidizing enzyme, theoxidizing enzymes within the sandwich complexes and/or competitivebinding complexes of each localized sample oxidize the substratemolecules present in the aqueous media used to transport said substratemolecules. In embodiments in which sandwich complexes are present,oxidation occurs at an electrochemical cell formed by an electrochemicalsensor and the volume of liquid substantially over it, and electronsflow from the working electrode of the electrochemical sensor to thevolume substantially above said sensor in a quantity proportional to aquantity of target analyte present within the localized sample. Inembodiments in which competitive binding complexes are present,oxidation occurs at an electrochemical cell formed by an electrochemicalsensor and the volume of liquid substantially over said sensor, andelectrons flow from the working electrode of the electrochemical sensorin a quantity inversely proportional to a quantity of target analytepresent within the localized sample.

At block 3218, the reader's processor receives from the reader'selectric connector a first signal detected at the electrochemicalsensor. In various embodiments, the signal is a voltage or currentsignal. At least a portion of the signal is caused by the oxidation ofthe substrate. At block 3220, the reader's processor receives from thereader's electric connector a second signal detected by a referencesensor. At block 3222, the reader's processor calculates a resultantsignal by subtracting or applying another algorithm to remove the secondsignal from the first signal to account for and/or eliminate noise thatmay be present within the system. At block 3224, the reader's processorprocesses and analyzes the resultant signal to identify the presenceand/or quantity of a target analyte. Optionally, as shown at block 3226,in some embodiments, the reader transmits signals indicative of a testresult to a remote computing device for further processing, storage,transmission to a server, and/or display of results to a user.

Some embodiments of the analyte detection system include a graphicaluser interface (GUI) configured to present results and other data to auser in a meaningful and easy-to-interpret manner. The GUI may begenerated by, and form part of, a software application accessible via aremote computing device as a downloadable application or through aninternet browser. As in FIGS. 28A and 28B, the remote computing devicemay be a mobile computing device, such as, for example, an iPhone orother smartphone, a tablet, or wearable device, or a laptop or othercomputer.

Due to the sensitive nature of the health-related information generatedby the system, in some embodiments, a user must log into the applicationupon every use. Logging in may include providing a username andpassword, which the application compares to a database of registeredusernames and passwords. If the entered username and password match anentry in the database, the user will be allowed to access the contentavailable within the application. In other embodiments, biometricrecognition technology may be used to log a user into the application.

In various embodiments, the user may enter personal information into aprofile using the GUI, and the application directs the remote computingdevice to transmit the entered information to a server for storage. Theprofile may include biographical information, such as, for example, thename, username, password, age, sex, race, address, genotyping data,height, and/or weight of the user. In some embodiments, some or all ofthis information may be pulled by the application from another socialnetwork to which the user links, such as, for example, Facebook,Google+, LinkedIn, Foursquare, or the like. In some embodiments, a usermay be able to save additional healthcare-related information into theprofile such as the name and contact information of the user's primarycare physician and/or the name and contact information of the user'spreferred pharmacy. The profile may also include a patient's medicalhistory, which in some embodiments, is dynamically and automaticallyupdated when health data is received by the remote computing device fromthe reader and subsequently transmitted to the server.

In some embodiments, the remote computing device receives dataindicative of a test result from the reader. Upon receiving the data,the application may direct the remote computing device to transmit thisdata to a server (e.g., an application server, database server, etc.)for storage within a database and request and receive additionalinformation from the server. For example, in some embodiments, thereader transmits data indicative of a test result to the remotecomputing device. The remote computing device may send the current testresult to the server for storage; the application may also direct theremote computing device to request and receive, from the server, pasttest results associated with the same patient and the same test type.Such information may be stored within a database of the server. Uponreceiving the past and present test results, the application of someembodiments generates a graph or table within the GUI of the remotecomputing device showing the results over time or changes in the resultsover time. In other embodiments, all data may be stored locally on theremote computing device such that no server is necessary. In suchembodiments, when the remote computing device receives data indicativeof a test result from a reader, the application directs the remotecomputing device to store the data locally and may further direct theremote computing device to analyze the data and display it visuallywithin the GUI. Some non-limiting example graphs and accompanyinginformation displayed by various GUIs are provided in FIGS. 33-38.

As shown in FIGS. 33-38, upon receiving the data indicative of a testresult, the application may also direct the remote computing device torequest, from the server, relevant information from a user's profile, orif data is stored locally, the application may pull relevant informationfrom a user's profile stored within memory. In some embodiments, theapplication identifies one or more actions, for the remote computingdevice or the user to take, based on the test results and the user'sprofile.

For example, as shown in FIG. 33, upon receiving data indicative of atest result regarding inflammation levels for a user, the remotecomputing device requested and received test results of the user'sinflammation levels over the past week; said test results are displayedwithin the GUI as a line graph depicting the trend in levels. The GUIalso includes a red zone providing a clear indication of when a testresult is within an undesired, elevated range. When elevated, theapplication may also generate, and the GUI may display, recommendationsfor user action, such as, for example, recommending that the user:“Recover with a green smoothie.”

As shown in FIG. 33, the application of some embodiments can also pulldata from other applications stored on the remote computing deviceand/or from other wireless wearable technologies. For example, sleepdata and exercise data may be pulled from a wearable band such as aFitbit® or iWatch® or from mobile health monitoring applications. TheGUI of some embodiments displays the test results from the reader alongwith information pulled from these other connected applications ordevices to provide the user with an integrated experience that allows auser to gain insights into why a result is occurring. For example, auser may be able to use the application to identify that inflammationlevels tend to rise immediately following a hard workout. In someembodiments, the application or a back-end program stored on the serverdoes the analysis, pulling in data from multiple sources, comparing thedata to known rule sets, and identifying correlations and trends.

Another example results screen from an exemplary GUI is provided in FIG.34. In FIG. 34, the remote computing device has received current testdata indicative of Vitamin D levels from the reader and pulled pastVitamin D level results from a database in a server. The GUI presentsseveral test results within a line graph to display trends in the dataand also provides a written summary, indicating that the user's VitaminD levels are falling and are down 15% since yesterday. The GUI alsopresents a recommendation for action, particularly, in this example, arecommendation to schedule activity outdoors where a user can absorbVitamin D from the sun. In at least some embodiments, the application isconfigured to: sync with a calendar application used by the user,identify the location of the user, check the weather forecast in theuser's location, and recommend particular times of day to go outside forsome sun.

Another example results screen from an exemplary GUI is provided in FIG.35. In FIG. 35, the remote computing device has received current testdata indicative of testosterone levels from the reader and pulled pasttestosterone level results from a database in a server. Similar to otherembodiments, the GUI may display, for example, a band showing a targetrange of testosterone levels, a line graph demonstrating where theuser's recent results fall relative to the target band, and a read outof the change since the last test result. Using the GUI or the GUI of aconnected application, a user may be able to track exercise, sleep, andfood consumption; information about such tracked data may be displayablein the presently provided GUI alongside the test results to provide amore holistic, comprehensive view of a user's wellness.

In various embodiments, the application may also be able to link to oneor more of a user's social networks. Additionally or alternatively,within the GUI, the user may be able to enter contact information forvarious people with which the user may wish to share test results. Forexample, as shown in FIG. 36, the depicted GUI is displaying testresults for a target analyte indicative of fertility levels. In thedepicted example, the user's fertility is peaking. A user may wish toshare this alert with her partner. Accordingly, a share icon isprovided, which when selected, allows the user to select one or morecontacts with whom to share the information. The user may be providedwith options to share the information via a text message, email, orprivate message, or if the contacts are also members of thesystem/network provided herein, the user may send a push notification orother alert within the application.

With other sensitive test results, a user may wish to share informationanonymously. In at least some embodiments of the present system, when atest result of a user may be relevant to others, the user can selectcontacts with whom the system will share an anonymous notification. Thenotification may alert the contact that someone in his or her networkreceived a positive test result for a particular test and/or recommendthat the contact get tested for a particular condition. If a user'sselected contacts are also members of the system/network providedherein, a push notification or other alert may be sent to the contact.If a user's selected contacts are not members of the network/system, theapplication may generate a text message, email, or private messagewithin a social networking site, to alert the contacts of therecommendation to get tested. The message may be sent by the applicationwithout providing any information about the user that prompted the alertto be sent. For example, if a user receives a positive test result for aparticular sexually transmitted infection, the user may enter contactinformation for past sexual partners that should be notified, and theapplication will message those individuals.

With other test results, a user may wish to share the results with aplurality of contacts within a social network. For example, is a childis found to have the flu or strep throat, a user such as a parent, maywish to share the information with everyone within his or her onlinesocial network or every parent within the child's class. One example isprovided in FIG. 37. The depicted GUI is displaying an example interfacefor a positive test result for the flu. As shown, the user may beprovided with options for sharing the test result with one or morepeople within the user's social network; for example, the user can sharethe test result by Facebook®, Twitter®, and/or email. The user may alsobe able to send a message, push notification, or alert to other membersof the network described herein.

As also shown in FIG. 37, in some embodiments, when a test yields apositive result for an infection or other medical condition, the usermay be provided with a prompt to contact a physician and/or to forwardthe results to a pharmacy and request a prescription. In someembodiments, if a user selects the option to contact a physician whileusing a smartphone, the system prompts the smartphone to dial the phonenumber of a physician stored within the user's profile. In someembodiments, when a positive test result for an infection or othermedical condition is generated, a user may be prompted to notify amedical provider (e.g., a physician, nurse practitioner, or pharmacist)who has joined as a member of the presently described network. If a userselects to share the result with an in-network medical provider, theapplication will share the result with the medical provider directlywithin the GUI of the provider's application. If the user's preferredmedical provider is not yet a member of the network, when a positivetest result is generated, the user may be prompted to invite his or hermedical provider to join the network. If the user provides an emailaddress of the medical provider, an electronic invitation will be sentvia the application.

In some embodiments, a user can invite a medical provider to join thenetwork, or if already a member, to connect via the network. Onceconnected, a user can elect to share selected data results or all theuser's data results with the medical provider. Similarly, in someembodiments, a user can invite a caregiver, family member, and/or friendto join the network, or if already a member, to connect via the network.Once connected, a user can elect to share selected data results or allthe user's data results with the caregiver, family member, and/orfriend. In this way, even remotely located caregivers can monitor thehealth of a patient and ensure the patient is performing recommendedtests regularly or as advised.

Additionally or alternatively, in some embodiments, the applicationenables users to track the prevalence and/or spread of a contagiousmedical condition on a map within the GUI. Such an example is shown inthe GUI of FIG. 37. In some embodiments, a pin or other marker mayappear on a map in the location of a positive test result; in otherembodiments, the map may be colored to depict the relative prevalence ofa condition by location. In other embodiments, other graphics may beused to present data regarding the spread of contagious illnesses.

It will be appreciated to those skilled in the art that any calculationsor other functions described herein as performed by the remote computingdevice may alternatively be performed by a remote server and transmittedto the remote computing device for display.

EXAMPLES

Various experiments have been performed to test and demonstrate theperformance of the system and components described herein.

Experiment 1

In a first set of experiments, the performance of variouselectrochemical sensors was tested. In particular, an electrochemicalsensor was formed having surface chemistry features consistent withembodiments described above. The electrochemical sensor had aself-assembled monolayer formed of mercaptoundecanoic acid andhexaethylene glycol dithiol (MUDA+HEG SAM). The signal-to-noise ratiogenerated by this electrochemical sensor was compared to thesignal-to-noise ratio of two controls: Control 1 was a bare sensor; andControl 2 was a sensor with a mercaptohexanol self-assembled monolayer(C6 SAM), which is a surface modification widely used in the industry toenhance the signal-to-noise ratio and stability of sensors. Each sensorwas positioned within a different analysis channel and the signal andnoise from each sensor were measured by a computing device electricallyconnected to the sensors. A target analyte, H3N2 nucleoprotein, wascombined both with magnetic particles bound to affinity agents and withdetector agents, forming sandwich complexes, and the sandwich complexeswere added to each analysis channel. Three trials were run across arange of H3N2 nucleoprotein concentrations. In particular, Trials 1, 2,and 3 used 4 ng/ml, 12 ng/ml, and 60 ng/ml of H3N2 nucleoprotein,respectively. Results of the trials are shown in FIG. 38. As shown, theelectrochemical sensor having a MUDA+HEG SAM surface chemistry greatlyoutperformed the control sensors across a range of concentrations.

Using the same test sensor and controls, the background noise of thesensors was also tested. Results from this test are provided in FIG. 39.As shown, the background noise and standard deviation of the sensorhaving a MUDA+HEG SAM surface chemistry were significantly reducedcompared to the controls. Reduced noise may lead to great increases inthe signal-to-noise ratio, as demonstrated in FIG. 38.

Using the same test sensor and Control 2, the shelf stability of thevarious surface chemistries was compared. In particular, the backgroundnoise was measured the same week the sensors were created, and again 2weeks, 1 month, 2 months, and 3 months later. Results from this test areprovided in FIG. 40. As shown, the background noise remainedconsistently lower with the sensor having a MUDA+HEG SAM surfacechemistry than with the sensor having the C6 SAM surface chemistry. TheC6 layer, which is widely used in industry, was shown to be unstableover time, with the performance degrading over the first two weeks.Accordingly, the MUDA+HEG SAM surface chemistry described herein wasshown to have significantly greater shelf-stability than the control.

Further tests were performed to study the signal and noise of theelectrochemical sensor. In one experiment, using an embodiment of thepresent system, a liquid carrying a chemical substrate,tetramethylbenzidine (TMB), localized over the sensor, and the sensorand liquid effectively formed an electrochemical cell. A constantpotential of approximately 200 mV was applied to the electrochemicalcell between the working and reference electrodes and the resultingelectric current was measured. Through several dozen measurementsperformed on different sensors having various surface chemistries, itwas found that the MUDA+HEG SAM sensors show a high degree of clusteringin the 10-30 nanoampere range, which is indicative of a very lowbackground noise relative to C6 SAM sensors and other tested sensors.See the measurement results provided in FIG. 41. This low backgroundnoise contributes to a high signal to noise ratio. In contrast to theconsistent, stable, and low signals generated from the MUDA+HEG SAMsensors, the other tested sensors often showed a high degree ofvariability. See the measurement results provided in FIG. 42. Forexample, with a bare sensor, the signals can be as high as 1-2 microampsand have variation up to a microamp. This leads to a high degree ofirreproducibility in an assay that uses bare sensors as the sensingcomponent.

Experiment 2

In a second set of experiments, the effect of including a sonicatorwithin the analyte detection system was tested. In particular, a testanalyte detection system and a control analyte detection system wereprovided. The test analyte detection system was made in accordance withthe present disclosure and included a sonicator disposed below the firstreservoir of the cartridge. The control system was similarly constructedbut without a sonicator. In both the test and the control system, asample was added to the first reservoir and allowed to remain there fortwo minutes to mix and hybridize with sample preparation reagents. Thesample added to the first reservoir included 60 ng/ml of the H3N2 virus.Sample preparation reagents for forming sandwich complexes with the H3N2virus were present within the first reservoir. In the test system, asonication protocol assisted with mixing the contents of the firstreservoir during the two minute time period. No sonication protocol wasapplied to the control system. After two minutes, the contents of thefirst reservoir was emptied into the analysis channel for detection andquantification of target analyte in the sample. The results of theexperiment are provided within FIG. 43. The presence of the sonicatorresulted in a substantial increase in the amount of captured targetbound within a sandwich complex, as indicated by an increase in signalat the electrochemical sensor. At the two minute mark, the test systemincreased the average signal 8-fold compared to the control system.Thus, the sonicator greatly increases the speed with which at least oneof the reactions necessary for target analyte detection occurs.

Experiment 3

A third set of experiments tested the system's effectiveness attransferring sample from the sample collection device to the firstreservoir. In a test system, formed in accordance with embodimentsprovided herein, a sample with a target analyte was provided on a firstend of a sample collection device, and the first end of the samplecollection device was inserted through an input tunnel into the firstreservoir. In a control system, formed in accordance with embodimentsprovided herein, the sample with target analyte was injected directlyinto the first reservoir using a pipette to avoid losing any sample. Inboth the test and control system, the sample then mixed with the samplepreparation reagents in the presence of a sonicator and sandwichcomplexes were formed between the target analyte and sample preparationreagents. After a designated period of time, a valve to the firstreservoir opened, and the sandwich complexes were washed into theanalysis channel where they localized over an electrochemical sensor andwere introduced to a chemical substrate. The signal from theelectrochemical sensor was recorded. It was found that signals in thetest system equaled 96.5% of the signal in the control system. Less than4% of the signal was lost in the transfer of target analytes from asample collection device into the first reservoir. In the future, such aloss may be accounted for in the quantification of the target analyte.Additionally, the loss is viewed as relatively insignificant compared tothe gains in usability that result from inserting a sample collectiondevice into the cartridge rather than injecting a sample into thecartridge via a pipette.

Experiment 4

A fourth set of experiments demonstrated the system's ability toreproducibly distinguish between differing concentrations of targetanalyte, and thus, demonstrated the system's ability to reproduciblyquantify target analytes. In one experiment, a target analyte,influenza, was inserted into the first reservoir where it mixed withinfluenza-specific sample preparation reagents and formed sandwichcomplexes. The sandwich complexes were washed into the analysis channelwhere they localized over an electrochemical sensor and were introducedto a chemical substrate. The signal from the electrochemical sensor wasrecorded. Multiple trials were run at five different target analyteconcentrations. In particular, the system was tested using: 120 ng/ml,60 ng/ml, 12 ng/ml, 4 ng/ml, and 0 ng/ml of influenza. Results of theexperiment are provided in FIG. 44. Both the average values and standarddeviations are shown. As depicted, the signals for each concentrationvalue differ and the signal decreases with decreasing concentrations oftarget analytes. The system demonstrated that it is capable ofgenerating data that will allow for the quantification of targetanalytes.

In another experiment, the same general experimental setup was used;however, instead of testing for influenza, the target analyte was CRP.Multiple trials were run at five different target analyteconcentrations. In particular, the system was tested using: 30 μg/ml, 15μg/ml, 5 μg/ml, 2 μg/ml, and 0.5 μg/ml (500 ng/ml) of CRP. Results ofthe experiment are provided in FIG. 45. Both the average values andstandard deviations are shown. As depicted, the standard deviations werevery small, showing good reproducibility of the measurements. Alsoshown, the signals demonstrate good linearity with a decreasing signalwith decreasing target analyte concentration. Accordingly, reproduciblequantification of the target analyte is possible with the system.

Experiment 5

A fifth set of experiments compared the system's time to result to thatof other known analyte detection systems. In testing for influenza, therun time from insertion of the sample collection device into thecartridge to receiving a final result took 3 minutes. In contrast, therun time for a standard ELISA assay is 120 minutes. Various innovationsincluded in embodiments of the system described herein lead to thisgreatly improved run time and contribute to an improved user experience.

Although the foregoing has included detailed descriptions of someembodiments by way of illustration and example, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof these embodiments that numerous changes and modifications may be madewithout departing from the spirit or scope of the appended claims.

What is claimed is:
 1. A system for generating a signal that detects atleast one of a presence, absence, and quantity of one or more analytes,the system comprising: a sample analysis cartridge comprising: an inputtunnel that extends from an aperture, the input tunnel configured topermit insertion of a distal portion of a sample collection deviceadapted to be exposed to a sample; a reservoir configured to hold aliquid and configured to receive the sample from the distal portion ofthe sample collection device; an analysis channel configured to receive,from the reservoir, the liquid having the sample and reagents mixedtherein; and an electrochemical sensor configured to be exposed to themixed liquid in the analysis channel and to generate a signal indicativeof at least one of the presence, absence, and quantity of one or moreanalytes within the sample, the electrochemical sensor comprising amixture of an oligo-ethyleneglycol dithiol and a carboxylic acidterminated thiol.
 2. The system of claim 1, further comprising a sampleanalysis reader electrically coupled to the sample analysis cartridge,the sample analysis reader configured to receive the signal forprocessing and to transmit the processed signal indicative of at leastone of the presence, absence, and quantity of the one or more analyteswithin the sample to a computer; and a computer readable medium withinstructions that, when executed by a processor of the computer, cause agraphical user interface to display information indicative of at leastone of the presence, absence, and quantity of the one or more analytes.3. The system of claim 1, wherein an oligo-ethylene glycol dithiol isselected from hexa-ethylene glycol diothiol and tetra-ethylene glycoldithiol.
 4. The system of claim 1, wherein the carboxylic acidterminated thiol is 11-mercaptoundecanoic acid.
 5. The system of claim2, wherein the graphical user interface is generated by an applicationaccessible via the computer.
 6. The system of claim 5, wherein theapplication is downloadable from the Internet.
 7. The system of claim 5,wherein the application requires a secure login prior to use.
 8. Thesystem of claim 2, wherein the computer is configured to transmit andreceive data to and from a server.
 9. The system of claim 8, wherein thedata comprises data indicative of at least one of the presence, absence,and quantity of the one or more analytes.
 10. The system of claim 8,wherein the data comprises past test results stored at the server. 11.The system of claim 2, wherein the information displayed on thegraphical user interface comprises a recommendation for user actionbased on at least one of the presence, absence, and quantity of the oneor more analytes.
 12. The system of claim 2, wherein the informationdisplayed on the graphical user interface comprises a table or graph orboth based on at least one of the presence, absence, and quantity of theone or more analytes.
 13. The system of claim 2, wherein the computer isconfigured to receive data from a wearable device, and wherein theprocessor of the computer is configured to process the data from thewearable device and to cause the graphical user interface to displayinformation based on the data from the wearable device.
 14. The systemof claim 5, wherein the application is configured to sync with acalendar application and to notify a user at particular times based onthe synchronization with the calendar application.
 15. The system ofclaim 2, wherein the application is configured to link to one or more ofa user's social networks.
 16. The system of claim 15, wherein theapplication is configured to notify one or more contacts in the user'sone or more social networks of test results indicative of at least oneof the presence, absence, and quantity of the one or more analyteswithin the sample.
 17. The system of claim 2, wherein the application isconfigured to notify the one or more contacts about test resultsindicative of fertility levels.
 18. The system of claim 15, wherein theapplication is configured to anonymously notify the one more contacts inthe user's social networks.
 19. The system of claim 2, wherein theinstructions, when executed by the processor, cause the graphical userinterface to prompt a user to contact a physician or pharmacy or bothbased on at least one of the presence, absence, and quantity of the oneor more analytes.
 20. The system of claim 2, wherein the graphical userinterface is configured to display a map indicating positive testresults for one or more analytes by other users at locations on the map.21. The system of claim 2, wherein the computer comprises a smartphone,a tablet, a wearable device, or a laptop.
 22. The system of claim 2,wherein receipt of the sample analysis cartridge by the sample analysisreader causes electric coupling between the sample analysis cartridgeand the sample analysis reader.
 23. The system of claim 1 or 2, whereinthe system further comprises the sample collection device.